Methods for treating and diagnosing blinding eye diseases

ABSTRACT

This invention relates to, in part, methods and compositions that are useful for the diagnosis, treatment, or prevention of a blinding eye disease, including in the discovery of drugs that are efficacious against these diseases. Diseases include, for example, age related macular degeneration and reticular pseudodrusen disease, and the methods described herein include, for example, the method named delayed near infrared analysis (DNIRA).

PRIORITY

This application is a continuation of U.S. patent application Ser. No.13/838,473, filed Mar. 15, 2013, which claims priority to and thebenefit of U.S. Provisional Patent Application No. 61/693,226, filedAug. 24, 2012, U.S. Provisional Patent Application No. 61/641,393, filedMay 2, 2012, and U.S. Provisional Patent Application No. 61/640,854,filed May 1, 2012, each of which is incorporated herein in theirentireties by reference.

FIELD OF THE INVENTION

This invention relates to methods and compositions that are useful forthe diagnosis, treatment, or prevention of a blinding eye disease,including the discovery of drugs that are efficacious against thesediseases.

BACKGROUND

Blinding eye diseases include a number of disorders that effect vision.Age-related macular degeneration (AMD) is the leading cause of oculardysfunction, including blindness. One form of AMD, non-exudative AMD,also known as “dry” AMD, is typically characterized by drusenaccumulation within or external to the retinal pigment epithelium (RPE).Late in dry AMD, atrophy of the RPE and the overlying rod and conephotoreceptors occurs. A second form of AMD, exudative or “wet” or“neovascular” AMD, is characterized by choroidal neovascularization.Early stage disease is frequently dry AMD while later stage disease isfrequently either wet (neovascular) AMD or dry (atrophic) AMD.

Drusen are whitish spots or deposits that occur within the RPE orexternal to it. Drusen are the defining or pathognomic feature of AMD.By contrast, the terms “pseudodrusen” or “drusenoid deposits” or“subretinal drusenoid deposits” have been used to describe discretedeposits or a yellowish curvilinear or reticular pattern that aresuggested to lie anterior to the RPE, relative to the path of lightentering the eye, in the sub-retinal space.

Because of differences in imaging techniques, there is controversyaround the prevalence of reticular pseudodrusen (RPD), with manyhypothesizing that it is under-estimated. Like AMD, RPD is associatedwith aging. RPD is also strongly associated with vision loss, typicallydue to geographic atrophy or choroidal neovascularization. Unlikeclassic AMD, it is however characterized by an interlacing, curvilinearor reticular pattern that can be visualized in multiple wavelengths oflight including but not limited to white, blue, blue autofluorescence,red-free, near infra-red or infra-red. The defining or pathognomicfeature of RPD is not drusen but rather “pseudodrusen”, “drusenoiddeposits” or “subretinal drusenoid deposits” that appear as base-downdomes, triangles or spike-like deposits that lie anterior to (above) theRPE in the subretinal space, when evaluating the outer retina and RPE inthe transverse plane, as can occur optically using for example, opticalcoherence tomography (OCT) or in post-enucleation samples using forexample, histology with or without immunohistochemistry. Unlike classicAMD, RPD may be associated with increased cardiovascular death. Atpresent, without wishing to be bound by theory, RPD may be considered adistinct form of age-related macular disease rather than a subtype ofAMD. RPD may also be considered to be a “diffuse-trickling” subtype ofdry AMD.

There is presently a paucity of drugs for effective treatment ofblinding eye diseases, such as AMD or RPD. Therefore, there remains aneed for therapies that are useful for treating these diseases and otherblinding eye diseases. Further, there is a need for effective diagnosisof these diseases.

SUMMARY OF THE INVENTION

Accordingly, in one aspect, the invention provides a method foridentifying whether a candidate compound is useful for the treatment ofa blinding eye disease, comprising (a) administering an effective amountof a test compound to an animal whose eye comprises (i) a fluorescentcompound in an amount effective to indicate the presence of a blindingeye disease in the animal and (ii) a toxin in an amount effective toinduce atrophy of ocular tissue; (b) exposing the eye to light having awavelength and intensity effective to cause the fluorescent compound tofluoresce; (c) comparing the eye's fluorescence pattern to afluorescence pattern of an animal's eye that comprises the fluorescentcompound and the toxin but not the test compound; and (d) selecting thetest compound as a candidate compound if the result of the comparison ofstep (c) indicates that the test compound is useful for the treatment ofa blinding eye disease.

In another aspect, the invention provides a method for treating orpreventing dry AMD, comprising administering to a subject in needthereof an effective amount of a compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein each of R₁ and R₂is independently H or a C₁-C₆ alkyl and R₃ is H or a C₁-C₆ alkyl.

In a further aspect, the invention provides a method for treating orpreventing dry AMD, comprising administering to a subject in needthereof an effective amount of methotrexate or a pharmaceuticallyacceptable salt thereof.

In another aspect, the invention provides a method of treating RPDdisease, comprising administering to a subject in need thereof aneffective amount of a compound of Formula I (as described herein) or apharmaceutically acceptable salt thereof, wherein each of R₁ and R₂ isindependently H or a C₁-C₆ alkyl and R₃ is H or a C₁-C₆ alkyl.

In still another aspect, the invention provides a method of treating RPDdisease, comprising administering to a subject in need thereof aneffective amount of methotrexate or a pharmaceutically acceptable saltthereof.

In yet another aspect, the invention provides a method for identifying asubject who has a blinding eye disease and is likely to respond totreatment with an agent comprising determining whether the subject's eyehas, or previously had, an increase (including a transient increase) inpermeability across the epithelial barrier between a choroid and aretina of the eye relative to an undiseased state; wherein the increasein permeability indicates that the subject is more likely than not torespond to treatment with the agent; and wherein the agent is selectedfrom methotrexate or a pharmaceutically acceptable salt thereof, or anda compound of Formula I (as described herein) or a pharmaceuticallyacceptable salt thereof, wherein each of R₁ and R₂ is independently H ora C₁-C₆ alkyl and R₃ is H or a C₁-C₆ alkyl.

In still another aspect, the present invention provides a method foridentifying a blinding eye disease subject who is more likely than notto respond to treatment with an agent comprising determining whether thesubject's eye has an presence (e.g. an influx) of phagocytic immunecells across a RPE relative to an undiseased state, wherein the presenceof phagocytic immune cells indicates that the subject is more likelythan not to respond to treatment with the agent; and wherein the agentis selected from methotrexate or a pharmaceutically acceptable saltthereof, or and a compound of Formula I (as described herein) or apharmaceutically acceptable salt thereof, wherein each of R₁ and R₂ isindependently H or a C₁-C₆ alkyl and R₃ is H or a C₁-C₆ alkyl.

In another aspect, the invention provides a method for determiningwhether a blinding eye disease in a subject is responsive to treatmentwith an agent that inhibits the function of a subject's immune cells,comprising detecting a presence, detecting an absence, or measuring anamount of immune cells in the subject's eye, wherein the subject's eyefluoresces in response to light having a wavelength of about 600 nm toabout 900 nm.

The details of the invention are set forth in the accompanyingdescription below. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, illustrative methods and materials are now described.Other features, objects, and advantages of the invention will beapparent from the description and from the claims. In the specificationand the appended claims, the singular forms also include the pluralunless the context clearly dictates otherwise. Unless defined otherwise,all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-D show that in vivo fundus autofluorescence (FAF) imagingfollowing NaIO₃ injection identifies geographic areas of damage thatdevelop a reticular pattern. ICG angiography identifies transientchorioretinal permeability.

FIG. 1A shows a time course of early FAF change around the optic nervehead. A hyperfluorescent line or border formed 2-3 days after NaIO₃injection. The border may or may not completely surround the optic nervehead, and is not contiguous with it. A lacy or reticular pattern offluorescence appeared between days 3 and 7, and lied peripheral to thisline. By day 7 this pattern was distinct.

FIG. 1B shows composite images that include the optic nerve head andmid-peripheral retina and illustrate that the hyperfluorescent bordersdefined areas of future lacy or reticular hyperfluorescence. In thisexample, the borders seen at Day 3 post-NaIO₃ defined an island in theinferior retina. By day 7 the border was no longer visible, and instead,profound reticular patterns emerged. Note that the image showing twodistinct islands at Days 3 and 7 are from different animals anddemonstrate how consistently the patterns is observed. Small islandssuch as these were seen in about 7% of eyes.

FIG. 1C shows that the pattern of FAF is distinctly different from theretinal and choroidal vasculature patterns seen on fluorescein (left andmiddle image of the inner and mid-retina, respectively) or ICGangiography (right image) of the choroid

FIG. 1D shows a transient increase in trans-epithelial permeability thatoccurred around Day 2 before pattern progression and is evident by asignificant flush during ICG angiography (790 nm, approximately 6minutes after dye injection). This ICG flush was not detected on Day 1following toxin injection, appeared briefly, and is rarely seen by thenext time point routinely tested, at Day3 or 4.

FIGS. 2A-D show maturation of FAF change after NaIO₃ injection in large360° rings.

FIG. 2A shows large composite FAF images encompassing much of theposterior pole and identified a 360° ring of damage defined by both aperi-papillary and peripheral retinal border. FAF images taken 7 daysafter NaIO₃ identified multiple discrete and contiguous areas ofcurvilinear hyperfluorescence that form oval, round, scalloped, rosette,or paw-print patterns. These typically emerged between days 7 and 14.Detail is provided in the upper enlargement. These complex FAF patternsarise within the earlier hyperfluorescent borders, and coalesce overtime.

FIG. 2B shows that by day 14 most animals show a near contiguous patternof reticular hyperfluorescence throughout the entire 3600 ring. Detailis provided in the middle box.

FIG. 2C shows that by 28 days after NaIO₃ exposure, the pattern took ona more granular appearance. Small areas where the pattern is lessapparent are seen as darker, grey areas.

FIG. 2D shows a clinical image of the “diffuse trickling” pattern of FAFfor comparison. The present invention, inter alia, recapitulates many ofthe features including reticular, lobular or scalloped ovals of greyhypofluorescence associated with peripheral hyperfluorescence.

FIGS. 3A-C show that a curvilinear, reticular pattern of outer retinaldeformation develops in the weeks after NaIO₃ injection.

FIG. 3A shows that this pattern corresponds with the distribution ofautofluorescent cells. Low power (1×, left column; 5×, right column)white light images of excised retinal whole mounts taken prior to NaIO₃injection (baseline), and at days 4 and 7 post-injection are shown. Asubtle curvilinear pattern is noted 3-4 days after compared to baseline,and is apparent at day 7.

FIG. 3B shows that by day 14 post-NaIO₃, this reticular pattern ispronounced and corresponds with gross deformation of the outer retina.This appeared as notches, or folds, in cross-sections seen at the siteof relaxing incisions (enlarged in box).

FIG. 3C shows that epifluorescent microscopy in the 488 nm channel, withno cellular staining, showed the presence of autofluorescent cells thatlie within the reticular grooves. This was evident both using broadlight emission (left column) as well as narrow excitation and emission(right column) and corresponds to the wavelength used during FAFimaging. Folds in the retina were associated with the presence of smallfluorescent dots by Day 7. Further enlargement clearly showed thedistribution of small fluorescent cells, with voids for their nuclei.Positive control tissue following systemic fluorescein-dextran injection(as for angiography) confirmed the ability to detect 488 nm fluorescencein excised retina or eyecup (similar in wavelength to that used for FAFin HRAII imaging) without tissue processing or staining. Negativecontrol at baseline shows absence of autofluorescent cells.

FIGS. 4A-C show interpretative illustrations which show howhistologically-determined tissue findings can account for in vivoimaging following NaIO₃-induced RPE loss.

FIG. 4A shows clinical images from a patient with early AMD and showsreticular pseudodrusen and so-called “target” lesions in the NIR channel(top). Immediately below are two schematic overlays that emphasize thehypofluorescent spots (those typically defined as drusenoid deposits)first in black for emphasis, and then with a bright background toemphasis the intervening bright signal. The bottom two images highlightmultiple small target lesions of RPD (within the circles), while thebottom-most schematic, illustrates the presence of a potential halo andtarget lesion formed by concentric folds of tissue.

FIG. 4B shows schematic renderings which indicate a direct relationshipbetween three-dimensional whole mount ONL histology and two-dimensionalen face clinical FAF images. By day 14 after NaIO₃ exposure, the outerretina shows complex folds of the mid-ONL (shown at relative lowmagnification [original 40×] and digitally enlarged). Digitalmagnification shows concentric rings of tissue folds. This same areaillustrated in black and white shows how this might appear on FAF to bea circular or oval region with a central void. Reversed black and whiterendering (a “negative” image), shows that this arrangement of ONLdistortion would appear to have a central bright target.

FIG. 4C shows OCT imaging of the rat retina 14 days after NaIO₃injection clearly shows bright “spikes” through the outer retina. Theseare described clinically. Similar histological sections through theouter retina demonstrate ONL folds with underlying inflammatory cells inthe enlarged subretinal space that form narrow columns of cells. Forcomparison with in vivo images, these are rendered in black and white.In reversed black and white (a pseudo-negative image to match OCTimages), it is seen that the ONL disappears while the subretinaldensities appear bright.

FIGS. 5A-H show that speckled NIR fluorescence is detected by 795/810 nmcSLO imaging, but cannot be detected without previous ICG injection,i.e., this fluorescence can only be detected with theexcitation/emission filters in place, but is not coincident with or inthe transit period following dye injection (that is, using the DNIRAmethod). FIG. 5A-F show representative normal confocal scanning laserophthalmoscopy (cSLO) and angiographic images of wild-type SD ratsobtained using:

FIG. 5A: red free.

FIG. 5B: infrared reflectance (830 nm).

FIG. 5C: 488/500 nm autofluorescence (FAF).

FIG. 5D: 795/810 nm NIR fluorescence.

FIGS. 5E-F illustrate normal retinal and choroidal vasculature usingfluorescein dextran and ICG dye, respectively. These and similar imagesserved as normal control for all experiments. Such imaging is used toproperly align the depth of focus within and between the retina and RPElayers.

FIG. 5G shows a time course of delayed near infrared analysis (DNIRA) ina representative animal that received a single injection of 5 mg/kg ICGdye at t=0. No detectable signal is seen prior to dye injection (notehomogenously dark image, upper row). During angiography (second row) theretinal blood vessels were visualized, and background was not detectablenoting that the gain (sensitivity) of the cSLO is reduced to preventsaturation during the transit phase. In the days after angiography,(rows 3-5) there is an increased speckled or punctate backgroundfluorescence compared to baseline. The delayed NIR fluorescence isblocked by overlying blood vessels and is absent at the optic nervehead, but unlike FAF (FIG. 5H) forms a bright arc or ring around thenerve head. The signal remains strong and distributed throughout thefundus out to 28 days after injection at the concentration used (5mg/kg). The slightly darker segment of the bottom row likely reflectsthe presence of a choroidal vortex vein.

FIG. 5H shows, in contrast to the DNIRA time course, the 488/500 nmautofluorescence signal reverts to pre-injection intensity (top row)when viewed at 3, 8 and 28 days after fluorescein angiography (lowerrows). The slightly darker segment of the bottom row likely reflects thepresence of a choroidal vortex vein.

FIGS. 6A-D show that the intensity of NIR fluorescent signal correlateswith ICG dosage using DNIRA.

FIG. 6A shows control cSLO images illustrate an absence of 795/810 nmfluorescent signal when no ICG dye injection has been given. Controlanimals that did not receive ICG maintained the same level of backgroundfluorescence with negligible signal over the 21 day period evaluated.

FIG. 6B show that using the same gain, delayed NIR fluorescence wasreadily detectable 48 hours after ICG injection when given at the lowdose of 0.35 mg/kg. However, with time, DNIRA shows gradual fading from48 hours to 21 days post-injection.

FIG. 6C show that using the same gain, 5 mg/kg ICG angiography resultedin brighter (higher intensity) delayed NIR fluorescence at 48 hours thandoes the lower dose (FIG. 6B). This fluorescence persisted largelyunchanged out to 21 days.

FIG. 6D shows that by contrast, the 830 nm NIR reflectance channel doesnot show similar change.

FIGS. 7A-B shows that sodium iodate causes patchy loss of delayed NIRfluorescence through which develops a clear view to the choroidalvasculature

FIG. 7A shows DNIRA performed 3 days after ICG and NaIO₃ injection. Lossof speckled fluorescence in clearly-defined patches or areas of theposterior pole was seen. These areas appeared dark, or hypofluorescent,with clearly defined borders.

FIG. 7B shows that within these areas, if the gain (sensitivity) of thecSLO is increased, the choroidal blood vessels were readily evident. Thebox (enlarged) identifies some of the prominent choroidal vessels thattravel in directions that are distinct from the retinal vasculature thatrun radially from the optic nerve (arrows). The speckled DNIRAfluorescence obscures the view to the choroid in the upper right of theimage.

FIGS. 8A-C show that the reticular FAF pattern matures in vivo and inexcised retina, and spares the area adjacent to the optic nerve.

FIG. 8A shows that despite significant outer retinal loss by month 3after NaIO₃ administration, the ONL was preserved in the areaimmediately adjacent to the optic nerve head (ONH). Higher magnification(bottom), confirmed that the photoreceptor nuclei are preserved, andthat the outer retina is not thrown into folds. FAF imaging of arepresentative eye from a Day 7 post-NaIO3 injection animal, confirmedthat the reticular pattern is not seen. Fine dotted line (inner circle)represents the location of the ONH, while the coarse dotted line (outercircle) delineates approximate extent of normal, non-reticular FAF.

FIG. 8B shows H&E staining which demonstrated clear deformation of theouter retina following NaIO₃ administration. Prior to NaIO₃administration (BL), the ONL appeared as a dark linear band thatextended from the ONH to the retinal periphery. By 7 days, the outerretina was thrown into folds but the ONL remained grossly intact. Thisis seen in the enlarged figure in the right column. However, by day 14post-NaIO3, the ONL showed areas of frank loss, with the inner retinalying directly against Bruch's Membrane (BM), the specialized basementmembrane of the RPE. This is seen in the enlarged figure (black arrow).

FIG. 8C shows confocal microscopy through the ONL imaged with thenuclear stain TO-PRO-3 (a carbocyanine monomer nucleic acid stain withfar-red fluorescence only, Invitrogen). At baseline, this layer wasflat, with no distinguishing features. With increasing time after NaIO₃administration, the ONL became progressively more deranged. Lineargrooves, curvilinear shapes, concentric rings, and isolated small ovalsor circles were identified at days 3, 6 and 14.

FIGS. 9A-C show that Iba1⁺ cells contribute to the reticular FAF patternin the absence of RPE early and late in disease.

FIG. 9A shows lower (top row) and higher (bottom row) magnification ofboth in vivo and ex vivo imaging which displays the comparison betweenthe reticular pattern seen on FAF and IHC early and late in diseaseprogression. The pattern of FAF identified in vivo is recapitulated byfluorescent immunohistochemistry using anti-Iba antibodies. Comparedagainst whole mount retina, excised immediately after HRAII imaging onDay 7, the same reticular pattern was seen using antibodies againstIba-1, a marker of microglia and macrophages (top). Nuclear stainingusing TO-PRO-3 showed densely packed cells of the outer nuclear layer(the photoreceptor layer) that appear, in two dimensions, to formcurvilinear, oval or lobular patterns. Merging of the two confirmed thatthe Iba-1⁺ staining is found interlaced between the nuclear staining.Enlargement of late stage reticular pattern confirmed that Iba-1⁺ cellsremained in the grooves between the folded photoreceptor nuclei(bottom).

FIG. 9B shows that FAF signal can be detected even in the absence ofRPE. Left: posterior eye cup, stained with RPE65 (green) shows loss ofthis monolayer in the central and mid retina (a ring of peripheral RPEremains. The remaining images utilized two different RPE labels (MiTF;microphthalmic transcription factor) and the characteristic doublenuclear stain, and confirms loss of the RPE layer in the mid and centraleyecup, in areas seen to have a complex pattern of fundusautofluorescence. By contrast, normal or near-normal RPE remains in theeyecup periphery.

FIG. 9C shows nuclear staining indicating the disappearance of RPE cellsin the central part of a whole mount retina as evident by the absence ofdouble nuclei which denote RPE cells. RPE was still visible inperipheral retinal tissue. This is in contrast to baseline control,where RPE cells were present in both central and peripheral retinaltissue.

FIGS. 10A-C show serial confocal microscopy through the outer retina,which displays a complex 3-dimensional deformation with correspondinginterlacing distribution of Iba-1⁺ and CD68⁺ phagocytic cells.

FIG. 10A shows morphological changes of the outer retina opticallysectioned into four layers extending from the inner plexiform layer(IPL) to the subretinal space. The ONL is divided into inner and outerlayers. Red=Iba-1, Green=CD68, Blue=nuclei. An increase in the number ofphagocytic cells was seen in the retinal layers at Day 4 after NaIO₃administration compared to baseline, at which time Iba-1 cells are foundprimarily in the inner retina. This was absent in antibody negativecontrols for both cell markers.

FIG. 10B shows that by day 14 after NaIO₃ administration, circular andoval patterns were evident in the ONL and as far internally as the OPL(identified by the presence of blood vessels). The pattern wascurvilinear in the inner ONL, and more so in the outer ONL where thetightly folded layer of nuclei are much more dense, with smaller areasof curvilinear voids. In the outer ONL, there was significant bridgingbetween folds. These nuclear voids contained Iba-1⁺ and CD68⁺ cells.

FIG. 10C shows an enlarged image in the outermost ONL or expandedsubretinal space (which is normally a hypothetical space) shows areticular pattern of Iba-1⁺ and CD68⁺ cells interlaced betweenphotoreceptor nuclei.

FIGS. 11A-B show three dimensional reconstruction of excised retinalwhole mount tissue which confirmed that phagocytic cells lie within thedeformed photoreceptor layer forming a reticular pattern

FIG. 11A shows confocal Z-series through the outer retina were used togenerate a three dimensional reconstruction of the ONL and Iba1⁺ cells.Nuclear staining (red) of the photoreceptor nuclei layer showed that itis thrown into folds. Merged with Iba-1⁺ staining (green), it wasevident that inflammatory cells lie between or within folds defined byretina deformation. Reconstruction of Iba-1 positive cells alonedemonstrated the reticular pattern.

FIG. 11B shows Z-series reconstruction of segments of the total imagestack of the outer retina 28 days after NaIO₃ administration illustratesthe complexity of the folds, that are more narrow at their peaks(inner), and broader and more interlacing at their bases (middle,outer). For comparison with cross-sectional imaging, and to betterrepresent the 3D nature of these findings, an oblique perspective isalso shown (middle panels). The position of the asterisk on the en faceimages (left) corresponded with the location of the asterisk in theoblique images. Digital enlargements (right) showed a groove between thephotoreceptor layer (red) that is curvilinear and contiguous in theouter retina, but broken into at least three smaller ovals in theinner-ONL. Iba1⁺ cells (green) were largely located within, i.e.external to, the photoreceptor layer. Control reconstruction of theouter retina showed neither Iba-1 positive cells nor deformation of theONL (bottom row). Bitplane software (Imaris) was used to generate theseimages.

FIGS. 12A-D show that progressive NaIO₃-induced outer retinaldeformation was identified by OCT imaging in vivo and compared toclinical findings (abbreviations: NFL=nerve fiber layer; GCL=ganglioncell layer; IPL=inner plexiform layer; INL=inner nuclear layer;OPL=outer plexiform layer; ONL=outer nuclear layer; IS+OS inner segment& outer segment layer; RPE=retinal pigment epithelium; BM=Bruch'smembrane).

FIG. 12A shows in vivo OCT and illustrates early changes that occurredin the inferior retina, but not the superior retina, were observed inthe first three days following NaIO₃ administration. Baseline imageindicated the retinal layers visible on OCT. En face image of the retinaat day 3 illustrated the position of the two optical sections, withsuperior and inferior portions shown. The superior portion was unchangedcompared to baseline. By contrast the inferior showed small undulationsof the photoreceptor inner and outer segments, along with loss of thethin RPE layer.

FIG. 12B shows that by Day 14 after NaIO₃ administration, the outerretina showed marked changes including the formation of multiple curvedfolds, base-down triangles and occasional spikes of bright signal thatlie external (inferior) to the dark outer nuclear layer.

FIG. 12C shows a well-formed base-down triangle is indicated in a day 14image of the rodent retina.

FIG. 12D shows OCT image of a patient with reticular pseudodrusenshowing a similar base-down triangle as that seen in the rodent model.

FIGS. 13A-B show that subretinal changes contributed to complex 3Dstructures in the Y-axis and Z-axis in vivo.

FIG. 13A shows adjacent, consecutive OCT images which confirm thatsubretinal changes contribute to complex 3D structures in the Y-axis.Small subretinal domes or pyramidal shapes are plentiful, and as showncentrally, also formed a shape similar to an upside down “Y” or“wishbone”. Compared against the volumetric reconstruction of serialimages this image coincides with optical sections through a structurethat appears circular when viewed enface. Images showing the position ofserial OCT sections through the circular structure correspond to thosein the adjacent optical cross-sections. The two major subretinal signalsof these cross-sections (arrows) are seen to be close together at theupper and lower extremities of the circle, and further apart in thecentre. The inter-spike distances are provided. Evaluation of thestructures immediately left of the circle show a similar contribution ofsubretinal structures to smaller loop-like structures.

FIG. 13B shows volumetric reconstructions of segmental stacks of serialZ-sections through the inner, mid, and outer ONL confirm the differenten face appearance of signal under the deformed outer retina. OCTsections through the same tissue plane. (Upper Row) Volumetricallyreconstructed images formed within the three planes bounded by theparallel green lines shown in upper row. Such segmental reconstructionthrough the inner, mid and outer-ONL demonstrates three distinctpatterns. (C. lower row) Regular array of punctate spots in the innerONL. (C, lower left) More curvilinear pattern in the mid-ONL. (C,middle) Outer-most ONL, largely within the expanded subretinal space,shows multiple complex curvilinear, looping, complex structures withsignificant bridging between them. These images correlate to the 3Dreconstructed confocal microscopy findings. (C, lower right)

FIGS. 14A-B show that gadolinium, which depletes circulating monocytesand resident macrophages alters the FAF pattern of NaIO₃-induced outerretinal damage.

FIG. 14A shows how, compared to untreated (bottom panel), GAD treatedlesion have less well-demarcated borders at Day 3 (top, left) and farless well demarcated patterns of FAF within the representative island ofdamage at Day (top, right).

FIG. 14B shows how macrophage depletion leads to smaller areas ofdamage. Left: F AF image shows small crescent of reticular pattern 7days after NaIO₃ and simultaneous GAD depletion of the macrophages.Right: Relative to the whole excised retina, the area of damage isclearly smaller that typically seen.

FIGS. 15A-C show interpretative illustrations of how, without wishing tobe bound by histologically-determined tissue findings can account for invivo imaging following NaIO3-induced RPE loss.

FIG. 15A shows clinical images from a patient with early AMD showingreticular pseudodrusen and so-called “target” lesions in the NIR channel(top). Immediately below are two schematic overlays that show thehypofluorescent spots (those typically defined as drusenoid deposits)first in black for emphasis, and then with a bright background toemphasis the intervening bright signal. The bottom two images highlightmultiple small target lesions of RPD (within the circles), while thebottom-most schematic, illustrates the presence of a potential halo andtarget lesion formed by concentric folds of tissue.

FIG. 15B shows schematic renderings suggest a direct relationshipbetween three-dimensional whole mount ONL histology and two-dimensionalen face clinical FAF images. By day 14 after NaIO₃ administration, theouter retina shows complex folds of the mid-ONL (shown at relative lowmagnification [original 40×] and digitally enlarged). Digitalmagnification shows concentric rings of tissue folds. This same areaillustrated in black and white showed how this might appear on FAF to bea circular or oval region with a central void. Reversed white & blackrendering (i.e., a “negative” image), showed that this arrangement ofONL distortion would appear to have a central bright target.

FIG. 15C shows OCT imaging of the rat retina 14 days after NaIO₃administration clearly shows bright “spikes” through the outer retina.These are described clinically. Similar histological sections throughthe outer retina demonstrate ONL folds with underlying inflammatorycells in the enlarged subretinal space that form narrow columns ofcells. For comparison with in vivo images, these are rendered in blackand white. In reversed black and white (i.e., a pseudo-negative image tomatch OCT images), it is seen that the ONL disappears while thesubretinal densities appear bright.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, of the discovery of a modalityfor detecting and/or measuring and/or evaluating a blinding eye diseasein a subject and/or an animal and the use of this method in thediscovery and/or evaluation of candidate compounds for improvedtreatments of the blinding eye diseases. Specifically, inter alia, thepresent invention provides for an analytical method entitled delayednear infra-red analysis (DNIRA), and the use of this method in thegeneration of models for a blinding eye disease such as, for example,AMD and/or RPD. Additionally, the present invention pertains to methodsof diagnosing specific forms of blinding eye disease, such as, forexample, AMD and/or RPD and methods for treating AMD and/or RPD.

In one aspect, the invention provides a method for identifying whether acandidate compound is useful for the treatment of a blinding eyedisease, comprising (a) administering an effective amount of a testcompound to an animal whose eye comprises (i) a fluorescent compound inan amount effective to indicate the presence of a blinding eye diseasein the animal and (ii) a toxin in an amount effective to induce atrophyof ocular tissue; (b) exposing the eye to light having a wavelength andintensity effective to cause the fluorescent compound to fluoresce; (c)comparing the eye's fluorescence pattern to a fluorescence pattern of ananimal's eye that comprises the fluorescent compound and the toxin butnot the test compound; and (d) selecting the test compound as acandidate compound if the result of the comparison of step (c) indicatesthat the test compound is useful for the treatment of a blinding eyedisease.

In some embodiments, the blinding eye disease is AMD or RPD disease.

In some embodiments, the animal is a mouse, rat, or zebrafish.

In other embodiments, the fluorescent compound absorbs light at awavelength of about 600 nm to about 900 nm and/or emits light at awavelength of about 750 nm to about 950 nm. In a specific embodiment,the fluorescent compound is ICG. In still other embodiments, thefluorescence occurs in RPE cells.

In some embodiments, the toxin is sodium iodate. In other embodiments,the atrophy comprises necrosis of RPE cells. In other embodiments, thenecrosis presents as patches.

In still other embodiments, the comparing occurs at least about 24hours, or at least about 7 days, or at least about 30 days, or at least60 days, or at least 90 days after administering the test compound.

In various embodiments, the exposing the eye to light comprisesperforming cSLO, FAF, DNIRA, or OCT. In various embodiments, theexposing the eye to light comprises white light, blue light, red-freelight, near infra-red, or infra-red.

In various embodiments, the presence of a blinding eye disease isindicated by patterns of FAF within patches of RPE damage or loss orouter retinal loss. In various embodiments, the presence of a blindingeye disease is indicated by patterns of FAF that occur within, oradjacent to, or in proximity to, or distant from, or in the absence ofpatches of RPE damage or loss or outer retinal loss. In someembodiments, the patterns are one or more of curvilinear, ribbon-like,reticular, oval, circular, scalloped, halo, and target-like lesions. Invarious embodiments, the presence of a blinding eye disease is indicatedby patterns of FAF within a border of patches of RPE damage or loss orouter retinal loss. In some embodiments, the patterns are one or more ofcurvilinear, ribbon-like, reticular, oval, circular, scalloped, halo,and target-like lesions. In various embodiments, the presence of ablinding eye disease is indicated by patterns of FAF that occur distantfrom, or in the absence of patches of RPE damage or loss or outerretinal loss. In some embodiments, the patterns are one or more ofcurvilinear, ribbon-like, reticular, oval, circular, scalloped, halo,and target-like lesions.

In various other embodiments, the presence of a blinding eye disease isindicated by cross-sectional patterns or transverse patterns. In someembodiments, the patterns are observed with OCT. In some embodiments,the patterns are RPE and/or outer retinal loss or mounds, triangles,peaks or spikes found in the sub-retinal space.

In still other embodiments, the methods further comprise the step ofobserving the eye prior to administering the test compound. In someembodiments, this observing establishes one or more pre-administrationcharacteristics of the eye.

In yet another embodiment, the methods described herein compriseadministering the fluorescent compound prior to administering the testcompound. In still another embodiment, the methods described herein donot comprise administering (i) an additional amount of a fluorescentcompound to the animal or (ii) a second fluorescent compound to theanimal. In other embodiments, the methods described herein compriseadministering the toxin prior to administering the test compound and/oradministering the toxin prior to administering the fluorescent compound.

In still another embodiment, the methods described herein compriseadministering (i) an additional amount of the toxin to the animal or(ii) a second toxin to the animal. In still another embodiment, themethods described herein comprise administering (i) an additional amountof the toxin to the animal, (ii) a second toxin to the animal, or (iii)a compound believed to influence the mechanism of action of the toxin.In some embodiments, the methods described herein comprise furthercomprise observing a reduction in the rate of formation, growth orexpansion of patches of ocular tissue atrophy or patches of tissue loss.

In some embodiments, the candidate compound is useful for treating,preventing, or reducing the rate of pathogenesis of a blinding eyedisease. In other embodiments, a plurality of candidate compounds isidentified. In some embodiments, the methods described herein furthercomprise comparing the usefulness of the plurality of candidatecompounds in the treatment of a blinding eye disease and selecting alead compound based on the comparison.

In another aspect, the invention provides a method for treating orpreventing dry AMD, comprising administering to a subject in needthereof an effective amount of a compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein each of R₁ and R₂is independently H or a C₁-C₆ alkyl and R₃ is H or a C₁-C₆ alkyl. In oneembodiment, the compound of Formula I is bindarit.

In a further aspect, the invention provides a method for treating orpreventing dry AMD, comprising administering to a subject in needthereof an effective amount of methotrexate or a pharmaceuticallyacceptable salt thereof.

In some embodiments, the methods further comprise administering anadditional therapeutic agent. In various embodiments, the additionaltherapeutic agent is one or more of an anti-vascular endothelial growthfactor (VEGF) agent, an angiotensin-converting enzyme (ACE) inhibitor, aperoxisome proliferator-activated receptor (PPAR)-gamma agonist orpartial agonist, or combined PPAR-alpha/gamma agonist, a renininhibitor, a steroid, and an agent that modulates autophagy.

In some embodiments, the dry AMD is identifiable by the presence ofareas of hyper-fluorescent FAF in an eye of the subject and/or thepresence of one or more areas of abnormally fluorescent FAF in an eye ofthe subject and/or by changes in one or more of blue spectrum fundusimaging, white-light fundus imaging, red-free fundus imaging, and OCT inan eye of the subject and/or by an increase (including a transientincrease) in permeability across the subject's epithelial barrierbetween a choroid and a retina relative to an undiseased state and/or bya deformation of the outer retina, and/or deformation of the mid-retinalvasculature across the subject's epithelial barrier between a choroidand a retina relative to an undiseased state and/or by the presence ofphagocytic immune cells across the subject's RPE relative to anundiseased state.

In some embodiments, the dry AMD is early stage AMD, or atrophic dryAMD.

In other embodiments, the subject is a human. In still otherembodiments, the administering is effected orally or intra-vascularly,or intraocularly, or periocularly, or to the ocular surface.

In another aspect, the invention provides a method of treating RPDdisease, comprising administering to a subject in need thereof aneffective amount of a compound of Formula I (as described herein) or apharmaceutically acceptable salt thereof, wherein each of R₁ and R₂ isindependently H or a C₁-C₆ alkyl and R₃ is H or a C₁-C₆ alkyl. In oneembodiment, the compound of Formula I is bindarit.

In still another aspect, the invention provides a method of treating RPDdisease, comprising administering to a subject in need thereof aneffective amount of methotrexate or a pharmaceutically acceptable saltthereof.

In some embodiments, the methods described herein comprise reducing theamount of pseudodrusen in the subject and/or reducing the amount ofpseudodrusen in any one of the foveal area, perifoveal area, juxtafovealarea, and extrafoveal area of the subject's eye. In other embodiments,the methods described herein comprise reducing the rates of progressionto late disease, wherein the late disease is any one of choroidalneovascularization or geographic atrophy. In some embodiments, themethods described herein comprise reducing the rates of expansion ofgeographic atrophy.

In some embodiments, the methods described herein comprise administeringan additional therapeutic agent. In various embodiments, the additionaltherapeutic is one or more of an anti-VEGF agent, an ACE inhibitor, aPPAR-gamma agonist or partial agonist, or combined PPAR-alpha/gammaagonist, a renin inhibitor, a steroid, and an agent that modulatesautophagy.

In still other embodiments, the RPD disease is identifiable by thepresence of one or more areas of distinct patterns of retinal imaging inthe eye of a subject, wherein the retinal imaging is one or more ofwhite light, red-free light, blue light, FAF, near infra-red (NIR),infra-red (IR), angiography, and DNIRA and/or the presence of one ormore areas of abnormally-fluorescent FAF in the eye of a subject and/oran increase (including a transient increase) in permeability across thesubject's epithelial barrier between a choroid and a retina relative toan undiseased state and/or a presence of phagocytic immune cells acrossthe subject's RPE relative to an undiseased state.

In other embodiments, the subject is a human. In still otherembodiments, the administering is effected orally or intra-vascularly,or intraocularly, or periocularly, or to the ocular surface.

In other embodiments, the invention provides for the use of compounds ofFormula I, methotrexate, or their pharmaceutically acceptable salts,alone or in combination with an additional therapeutic, in themanufacture of a medicament useful for the treatment or prevention ofone or more blinding eye diseases.

In yet another aspect, the invention provides a method for identifying asubject who has a blinding eye disease and is more likely than not torespond to treatment with an agent comprising determining whether thesubject's eye has, or previously had, an increase (including a transientincrease) in permeability across the epithelial barrier between achoroid and a retina of the eye relative to an undiseased state; whereinthe increase in permeability indicates that the subject is more likelythan not to respond to treatment with the agent; and wherein the agentis selected from methotrexate or a pharmaceutically acceptable saltthereof, or and a compound of Formula I (as described herein) or apharmaceutically acceptable salt thereof, wherein each of R₁ and R₂ isindependently H or a C₁-C₆ alkyl and R₃ is H or a C₁-C₆ alkyl.

In one embodiment, the compound of Formula I is bindarit. In someembodiments, the blinding eye disease is one or more of dry AMD and RPDdisease.

In still another aspect, the present invention provides a method foridentifying a blinding eye disease subject who is more likely than notto respond to treatment with an agent comprising determining whether thesubject's eye has a presence of phagocytic immune cells (optionallyderived from within the retina or from the RPE) across the RPE relativeto an undiseased state, wherein the presence of phagocytic immune cellsindicates that the subject is more likely than not to respond totreatment with the agent; and wherein the agent is selected frommethotrexate or a pharmaceutically acceptable salt thereof, or and acompound of Formula I (as described herein) or a pharmaceuticallyacceptable salt thereof, wherein each of R₁ and R₂ is independently H ora C₁-C₆ alkyl and R₃ is H or a C₁-C₆ alkyl.

In one embodiment, the compound of Formula I is bindarit. In someembodiments, the blinding eye disease is one or more of dry AMD and RPDdisease. In other embodiments, the presence of phagocytic immune cellsis measured by DNIRA.

In another aspect, the invention provides a method for determiningwhether a blinding eye disease in a subject is responsive to treatmentwith an agent that inhibits the function of a subject's immune cells,comprising detecting a presence, detecting an absence, or measuring anamount of immune cells in the subject's eye, wherein the subject's eyefluoresces in response to light having a wavelength of about 600 nm toabout 900 nm. In some embodiments, the light has a wavelength of about400 nm to about 900 nm

In some embodiments, the methods described herein further compriseadministering to the subject an effective amount of a fluorescentcompound, wherein the detecting or measuring occurs at least one dayafter the administration of the fluorescent compound. In someembodiments, the detecting or measuring occurs at least one day afteradministering to the subject an effective amount of a fluorescentcompound.

In some embodiments, the methods described herein further comprise thestep of detecting or measuring FAF in the eye of the subject. In someembodiments, the methods described herein further comprise the step ofcorrelating an FAF pattern to the presence, absence, or amount of immunecells in the subject's eye.

In other embodiments, the blinding eye disease is AMD, central serousretinopathy (CSR) or RPD disease. In some embodiments, the subject is ahuman.

In various embodiments, the subject's eye fluoresces light having awavelength of about 750 nm to about 950 nm. In some embodiments, thefluorescent compound is ICG.

In some embodiments, the detecting or measuring occurs at about one day,or about seven days, or at about thirty days after administration of thefluorescent compound.

In some embodiments, the methods described herein do not furthercomprise administering (a) an additional amount of the fluorescentcompound or (b) a second fluorescent compound.

In other embodiments, the detecting or measuring comprises performingcSLO, FAF, DNIRA or OCT.

In some embodiments, the immune cells are cells of the subject's innateimmune system and/or macrophage and/or microglial cells.

Definitions

The following definitions are used in connection with the inventiondisclosed herein. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood to one ofskill in the art to which this invention belongs.

An “effective amount,” when used in connection with a test compoundand/or candidate compound is an amount that is effective for providing ameasurable treatment, prevention, or reduction in the rate ofpathogenesis of a blinding eye disease such as, for example, AMD and/orRPD.

An “effective amount,” when used in connection with a fluorescentcompound is an amount that allows optical detection.

An “effective amount,” when used in connection with a compound ofFormula I, methotrexate, or a pharmaceutically acceptable salt thereof,is an amount that is effective for treating or preventing dry AMD and/orRPD disease (e.g. providing a measurable treatment, prevention, orreduction in the rate of pathogenesis).

An “effective amount,” when used in connection with another therapeuticagent is an amount that is effective for treating or preventing dry AMDand/or RPD disease (e.g. providing a measurable treatment, prevention,or reduction in the rate of pathogenesis) alone or in combination with acompound of Formula I, methotrexate, or a pharmaceutically acceptablesalt thereof. “In combination with” includes administration within thesame composition and via separate compositions; in the latter instance,the other therapeutic agent is effective for treating or preventing acondition during a time when the agent a compound of Formula I,methotrexate, or a pharmaceutically acceptable salt thereof, exerts itsprophylactic or therapeutic effect, or vice versa.

An “effective amount,” when used in connection with a toxin, forexample, sodium iodate, is an amount that is effective for inducingmeasurable atrophy of ocular tissue as described herein.

An agent is “useful for the treatment of a blinding eye disease” if theagent provides a measurable treatment, prevention, or reduction in therate of pathogenesis of a blinding eye disease.

The term “blinding eye disease” refers to one of various ophthalmicdiseases and includes diseases of the eye and the ocular adnexa. Theterm “blinding eye disease” includes, for example, disorders describedherein (for instance, by way of non-limiting example, AMD and RPD).

The term “neovascularization” refers to new blood vessel formation inabnormal tissue or in abnormal positions.

The term “VEGF” refers to a vascular endothelial growth factor thatinduces angiogenesis or an angiogenic process, including, but notlimited to, increased permeability. As used herein, the term “VEGF”includes the various subtypes of VEGF (also known as vascularpermeability factor (VPF) and VEGF-A) that arise by, e.g., alternativesplicing of the VEGF-A/VPF gene including VEGF₁₂₁, VEGF₁₆₅ and VEGF₁₈₉.Further, as used herein, the term “VEGF” includes VEGF-relatedangiogenic factors such as PIGF (placental growth factor), VEGF-B,VEGF-C, VEGF-D and VEGF-E, which act through a cognate VEFG receptor(i.e., VEGFR) to induce angiogenesis or an angiogenic process. The term“VEGF” includes any member of the class of growth factors that binds toa VEGF receptor such as VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), or VEGFR-3(FLT-4). The term “VEGF” can be used to refer to a “VEGF” polypeptide ora “VEGF” encoding gene or nucleic acid.

The term “anti-VEGF agent” refers to an agent that reduces, or inhibits,either partially or fully, the activity or production of a VEGF. Ananti-VEGF agent can directly or indirectly reduce or inhibit theactivity or production of a specific VEGF such as VEGF₁₆₅. Furthermore,“anti-VEGF agents” include agents that act on either a VEGF ligand orits cognate receptor so as to reduce or inhibit a VEGF-associatedreceptor signal. Non-limiting examples of “anti-VEGF agents” includeantisense molecules, ribozymes or RNAi that target a VEGF nucleic acid;anti-VEGF aptamers, anti-VEGF antibodies to VEGF itself or its receptor,or soluble VEGF receptor decoys that prevent binding of a VEGF to itscognate receptor; antisense molecules, ribozymes, or RNAi that target acognate VEGF receptor (VEGFR) nucleic acid; anti-VEGFR aptamers oranti-VEGFR antibodies that bind to a cognate VEGFR receptor; and VEGFRtyrosine kinase inhibitors.

The term “anti-RAS agent” or “anti-Renin Angiotensin System agent”refers to refers to an agent that reduces, or inhibits, either partiallyor fully, the activity or production of a molecule of the reninangiotensin system (RAS). Non-limiting examples of “anti-RAS” or“anti-Renin Angiotensin System” molecules are one or more of anangiotensin-converting enzyme (ACE) inhibitor, an angiotensin-receptorblocker, and a renin inhibitor.

The term “steroid” refers to compounds belonging to or related to thefollowing illustrative families of compounds: corticosteroids,mineralicosteroids, and sex steroids (including, for example,potentially androgenic or estrogenic or anti-androgenic andanti-estrogenic molecules). Included among these are, for example,prednisone, prednisolone, methyl-prednisolone, triamcinolone,fluocinolone, aldosterone, spironolactone, danazol (otherwise known asOPTINA), and others.

The terms “peroxisome proliferator-activated receptor gamma agent,” or“PPAR-γ agent,” or “PPARG agent,” or “PPAR-gamma agent” refers to agentswhich directly or indirectly act upon the peroxisomeproliferator-activated receptor. This agent may also influencePPAR-alpha, “PPARA” activity.

The term “an agent that modulates autophagy” refers to a modulator ofcell survival, cell death, survival, autophagy, proliferation,regeneration, and the like.

The term “monocyte chemotactic protein-1,” or “MCP-1” refers to a memberof the small inducible gene (SIG) family that plays a role in therecruitment of monocytes to sites of injury and infection.

The term “alkyl,” as used herein unless otherwise defined, refers to astraight or branched saturated group derived form the removal of ahydrogen atom from an alkane. Representative straight chain alkyl groupsinclude, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl,-n-pentyl, and n-hexyl. Representative branched alkyl groups include,but are not limited to, isopropyl, -sec-butyl, -isobutyl, -tert-butyl,-isopentyl, -neopentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl,1,1-dimethylpropyl and 1,2-dimethylpropyl.

As used herein, “a,” “an,” or “the” can mean one or more than one.Further, the term “about” when used in connection with a referencednumeric indication means the referenced numeric indication plus or minusup to 10% of that referenced numeric indication. For example, thelanguage “about 50” covers the range of 45 to 55.

As referred to herein, all compositional percentages are by weight ofthe total composition, unless otherwise specified. As used herein, theword “include,” and its variants, is intended to be non-limiting, suchthat recitation of items in a list is not to the exclusion of other likeitems that may also be useful in the materials, compositions, devices,and methods of this technology. Similarly, the terms “can” and “may” andtheir variants are intended to be non-limiting, such that recitationthat an embodiment can or may comprise certain elements or features doesnot exclude other embodiments of the present technology that do notcontain those elements or features.

Although the open-ended term “comprising,” as a synonym of terms such asincluding, containing, or having, is used herein to describe and claimthe invention, the present invention, or embodiments thereof, mayalternatively be described using alternative terms such as “consistingof” or “consisting essentially of.”

DNIRA

In various embodiments, the present invention involves optical imaging,using various techniques that are known in the art. For example, suchtechniques include, but are not limited to cSLO, FAF, angiography, OCT,including three dimensional reconstructions of such. In variousembodiments of the invention, exposing an eye to light comprisesperforming cSLO, FAF, DNIRA, angiography or OCT. In various embodiments,the imaging is DNIRA. In various embodiments, combinations of any of theabove techniques may be used.

For DNIRA, a compound suitable for fluorescence detection including anear-infrared (NIR) dye, such as, ICG when given at non-toxic doses, canlabel the RPE and therefore make it visible when viewed using the ICGexcitation/emission filters in the days or weeks thereafter.Importantly, this visualization in the days and weeks thereafter may bewithout re-administration of dye. Accordingly, in some aspects, acentral component of the DNIRA technique lies in its timing. This isdistinct from the present usage of ICG or other angiographic dyes thatare viewed immediately after injection, during the transit phase, or inthe immediate minutes to hours following injection, to determine theintra-vascular localization of dye and its immediate extravasation.

In one embodiment, DNIRA may involve administration of a compoundsuitable for fluorescence detection, by way of non-limiting example, ICG(and, optionally, angiography) at about one or more days prior toadministration with a toxin or other agent that causes geographicatrophy expansion (e.g. NaIO₃) and optionally followed by, at about 1 ormore days (or about one week, or about one month, or about threemonths), an additional amount of NaIO₃ or another agent that causesgeographic atrophy expansion. For example, the other challenge thatcauses geographic atrophy expansion (e.g. as an initial, or second, orthird, or fourth administration) may be a modulator of cell survival,cell death, survival, autophagy, proliferation, regeneration, and thelike.

Expansion of geographic atrophy is a U.S. Food and Drug Administration(FDA) approved primary outcome for clinical trial design, and,accordingly, this invention makes possible observation of geographicatrophy, in particularly the expansion of geographic atrophy, in ananimal model, thus permitting correlation between pre-clinical diseasemodels and clinical trial design. The inability to clearly identify thegeographic atrophy, or expansion of geographic atrophy, in an eye of ananimal prior to the present invention has precluded direct correlationbetween pre-clinical studies and clinical observation.

In some embodiments, the compound suitable for fluorescence detection issuitable for imaging with various wavelengths of fluorescence. In someembodiments, these wavelengths range from visible light to infrared,e.g., 390 nm to 1 mm, including, for example, blue light, white light,and near-infrared. In some embodiments, the dye is a near-infrared dye.In some embodiments, the dye is ICG.

In some embodiments, DNIRA is performed (and/or delayed near infraredfluorescence (DNIRF) is observed) at about 1 day, or about 2 days, orabout 3 days, or about 4 days, or about 5 days, or about 6 days, orabout 7 days, or about 10 days, or about 14 days, or about 21 day afterthe administration. In some embodiments, the DNIRA is performed at least1 day after the administration, or at least 2 days, or at least 3 days,or at least 4 days, or at least 5 days, or at least 6 days, or at least7 days, or at least 10 days, or at least 14 days, or at least 21 daysafter the administration. In other embodiments, the DNIRA is performedat least about 24 hours, or at least about 7 days, or at least about 30days, or at least 60 days, or at least 90 days after administering. Inother embodiments, the DNIRA is performed at least about 2 months, orabout 3 months, or about 4 months, or about 5 months, or at a maximumabout 6 months after administering. In some embodiments, the DNIRA isnot performed during the transit stage (i.e. the first passage of dye asit flows through the ocular blood vessels and into the ocular tissue) orminutes thereafter.

In some embodiments, the visualization is effected using a cSLO. In someembodiments, the visualization is effected using white light andappropriate filters. In some embodiments, the ICG excitation/emissionfilters are 795 nm (excitation)/810 nm (emission) filters. In someembodiments, the ICG excitation/emission filters are about 795 nm(excitation)/about 810 nm (emission) filters.

The RPE is a critical epithelial monolayer that serves a “nurse-cell”function for an eye's specialized photoreceptors, the rods and cones.Blinding eye diseases, such as, for example, AMD and RPD, are, withoutwishing to be bound by theory, causally linked in part to abnormalitiesof the RPE.

DNIRA makes it possible to clearly identify the RPE layer in vivo in aneye of an animal. Further, the leading technique used to detect the RPEin the human eye, FAF, is ineffective or poorly effective, possiblyowing to a relative paucity of fluorophores such as lipofuscin. FAFimaging in the human eye is performed using the blue spectrum ofnon-coherent light in the presence of stimulation/emission filters, orcoherent blue light, and can identify areas of absent RPE (e.g.hypo-fluorescent signal) or abnormal RPE (e.g. hyper-fluorescentsignal). The inability to clearly identify the RPE in an eye of ananimal, in the absence, has precluded direct correlation betweenpre-clinical studies and clinical observation.

Accordingly, in various aspects of the present invention, methods tomake visible the RPE layer, such as, for example, DNIRA, in an eye of ananimal for pre-clinical investigation of blinding eye diseases areprovided.

Further, as described herein, DNIRA, or variations thereof, allow forvisualization of fluorescent immune cells in the eyes of an animal. Insome embodiments DNIRA may optionally not comprise toxin administration.

Further, as described herein, DNIRA, or variations thereof, allow forvisualization of fluorescent immune cells in the eyes of human subject.In some embodiments with a human subject DNIRA may not comprise toxinadministration.

Blinding Eye Diseases

A blinding eye disease refers to one of various ophthalmic diseases andincludes diseases of the eye and the ocular adnexa.

In various embodiments, a blinding eye disease is evaluated and/oridentifiable using DNIRA or a technique known in the art. Illustrativetechniques include: cSLO, FAF, OCT (including with cross-sectional,three-dimensional and en face viewing), SD-OCT (with cross-sectional,three-dimensional and en face viewing), angiography, or other imagingmodalities including other wavelengths of fluorescence. In someembodiments, these wavelengths range from blue to infrared, e.g., 390 nmto 1 mm, including, for example, blue light, white light, red-free, nearinfra-red, infrared.

In various embodiments, a blinding eye disease is evaluated and/oridentifiable by patterns of FAF within patches of RPE damage or loss orouter retinal loss. In some embodiments, the patterns are one or more ofcurvilinear, ribbon-like, reticular, oval, circular, scalloped, halo,and target-like lesions.

In various embodiments, a blinding eye disease evaluated and/oridentifiable by patterns of FAF within a border of patches of RPE damageor loss or outer retinal loss. In some embodiments, the patterns are oneor more of curvilinear, ribbon-like, reticular, oval, circular,scalloped, halo, and target-like lesions.

In various other embodiments, a blinding eye disease is evaluated and/oridentifiable by cross-sectional patterns or transverse patterns. In someembodiments, the patterns are observed with OCT. In some embodiments,the patterns are RPE and/or outer retinal loss or mounds, triangles,peaks or spikes found in the sub-retinal space.

In other embodiments, a blinding eye disease is evaluated and/oridentifiable by the presence of areas of hyper-fluorescent FAF in an eyeof the subject and/or the presence of one or more areas of abnormallyfluorescent FAF in an eye of the subject and/or by changes in one ormore of blue spectrum fundus imaging, white-light fundus imaging,red-free fundus imaging, and OCT in an eye of the subject and/or by anincrease (e.g. a transient increase) in permeability across thesubject's epithelial barrier between a choroid and a retina relative toan undiseased state and/or by a deformation of the outer retina, and/ordeformation of the mid-retinal vasculature across the subject'sepithelial barrier between a choroid and a retina relative to anundiseased state and/or by the presence of phagocytic immune cellsacross the subject's RPE relative to an undiseased state. Illustrativechanges include differences in an imaging pattern in the same ofdifferent subject and/or animal at two different time points and/orexperimental or clinical conditions. For example, changes can encompasschanges in imaging data between two evaluations of the same subjectand/or animal and/or changes in imaging data between a first subjectand/or animal and the imaging data of a second subject and/or animal.

In still other embodiments, a blinding eye disease is evaluated and/oridentifiable by the presence of one or more areas of distinct patternsof retinal imaging in the eye of a subject, wherein the retinal imagingis one or more of white light, red-free light, blue light, FAF, nearinfra-red (NIR), infra-red (IR), angiography, and DNIRA and/or thepresence of one or more areas of abnormally-fluorescent FAF in the eyeof a subject and/or an increase (e.g. a transient increase) inpermeability across the subject's epithelial barrier between a choroidand a retina relative to an undiseased state and/or a presence ofphagocytic immune cells across the subject's RPE relative to anundiseased state.

In various embodiments, the blinding eye disease is AMD. AMD is theleading cause of blindness in the developed world. Early AMD ischaracterized by the accumulation of drusen, the hallmark of disease,while geographic atrophy (GA) and choroidal neovascularization (CNVM)are the blinding complications of late non-exudative (so-called “dry” oratrophic) and exudative (so-called “wet” or neovascular) disease,respectively. Anti-VEGF therapy has revolutionized treatment of wet AMD,but represents just 1²-15% of all AMD cases. Though vitaminsupplementation can slow the rates of progression no treatments existfor dry AMD.

In various embodiments, the blinding eye disease is early non-exudativeor “dry” AMD. Early non-exudative or “dry” AMD can be characterized bydrusen accumulation and associated features such as pigmentation change,or RPE detachments. Late, or advanced dry AMD can be characterized bypatchy atrophy of the RPE and overlying photoreceptors. These patchesare visualized clinically as so-called “window defects” and are known asareas of “geographic atrophy.”

In various embodiments, the blinding eye disease is wet AMD. Wet AMD ischaracterized by the growth of new blood vessels beneath the retina orRPE which can bleed and leak fluid, resulting in a rapid and oftensevere loss of central vision in the majority cases. This loss ofcentral vision adversely affects one's everyday life by impairing theability to read, drive and recognize faces. In some cases, the maculardegeneration progresses from the dry form to the wet form.

In one embodiment, the blinding eye disease is exudative or wet AMD. Inone embodiment, the blinding eye disease is associated with choroidalneovascularization (CNVM).

In some embodiments, AMD is detected or evaluated using FAF, an in vivoimaging method for the spatial mapping (e.g. two-dimensional, en face,and the like) of endogenous, naturally or pathologically occurringfluorophores of the ocular fundus. In some embodiments, without wishingto be bound by theory, FAF imaging allows visualization of the RPE invivo and can help to better understand its metabolic alterations in thepathogenesis of chorioretinal disorders and retinal pigmentepitheliopathies. FAF is well known in the art (see, e.g., Bellman etal. Br J Ophthalmol 2003 87: 1381-1386, the contents of which are herebyincorporated by reference).

In some embodiments, AMD and/or RPE damage or loss may also bevisualized by other wavelengths of light, including white light, bluelight, near infra-red, infra-red, and be visible as a “window defect”through which the choroid can be viewed. In some embodiments, AMD and/orRPE damage or loss may also be visualized by cSLO, FAF, OCT (includingwith cross-sectional, three-dimensional and en face viewing), SD-OCT(with cross-sectional, three-dimensional and en face viewing), or otherimaging modalities including other wavelengths of fluorescence (e.g.wavelengths ranging from blue to infrared, e.g., 390 nm to 1 mm,including, for example, blue light, white light, red-free, nearinfra-red, or infrared).

In some embodiments, in late dry AMD, areas of geographic atrophy areclearly visualized as areas of hypofluorescent, or dark, FAF. In someembodiments, these represent areas where RPE is lost.

In some embodiments, dry AMD is identifiable by the presence of areas ofhyper-fluorescent FAF in an eye of the subject. In some embodiments, thedry AMD identifiable by the presence of areas of hyper-fluorescent FAFis progressing early or late dry AMD. In some embodiments,hyper-fluorescent FAF refers to an increased fluorescence that may becaused by an enhanced visualization of a normal density of lipofuscin orlipofuscin-like materials or an increase in the lipofuscin content ofthe tissues. Lipofuscin comprises a mix of proteins, with or withoutlipid, the components of which fluoresce under blue-light illuminationof the appropriate wavelengths. Lipofuscin-like fluorescence may alsooccur in this spectrum, and could be due to molecules other thanlipofuscin and its constituents.

Lipofuscin-like fluorescence may occur in RPE cells, and cells otherthan RPE cells such as, for example, cells of the immune system (e.g.phagocytic immune cells).

In other embodiments, the AMD is identifiable by the presence ofabnormal patterns of FAF in an eye of the subject. In some embodiments,abnormal fluorescent FAF refers to deviation from the normal fluorescentFAF pattern observed in a subject's eye. In normal FAF, using the cSLOor modified fundus cameras, the optic nerve head is dark (black) due tothe absence of RPE (and hence no lipofuscin) and the blood vessels arealso dark because they block fluorescence from the underlying RPEmonolayer. In the central macular area, the FAF signal is reduced byabsorption of blue light by luteal pigment. These characteristics ofnormal blue-light FAF may be considered when evaluating for the presenceof abnormal fluorescence.

In some embodiments, hyperfluorescent FAF associated with AMD and otherblinding diseases, may show two-dimensional spatial patterns, which maybe complex. In some studies, these patterns of hyperfluorescent FAFcorrelate with rates of disease progression from early to late (eitherneovascular or atrophic) disease. These patterns are understood in theart (see, e.g., Schmitz-Valckenberg et al. Survey of Ophthalmology. 2009January-February; 54(1):96-117; Bindewald et al. British Journal ofOphthalmology. 2005 July; 89(7):874-8; Holz et al. Am. J Ophthalmology.2007 March; 143(3):463-72, the contents of which are hereby incorporatedby reference in their entireties).

In some embodiments, the dry AMD is early stage AMD, or atrophic dryAMD.

In some embodiments, dry AMD is in its late stage and characterizable bythe presence of areas of hyper-fluorescent or abnormally-fluorescent FAFin areas bordering and/or adjacent to (in the so-called junctional zone)pre-existent geographic atrophy.

In some embodiments, dry AMD is in its late stage and characterizable bythe presence of areas of hyper-fluorescent or abnormally-fluorescent FAFin the absence of pre-existent geographic atrophy. In these embodiments,without wishing to be bound by theory, it may predict future loss of theRPE.

In some embodiments, dry AMD in both early and late stage ischaracterizable by the presence of immune cells (e.g. phagocytic immunecells). The presence of immune cells can be surmised frompost-enucleation or post-mortem ocular samples. As described herein, insome embodiments, the presence of immune cells is assessed using DNIRA.

In some embodiments, patterns such as “diffuse trickling” may beevaluated, treated or prevented using methods disclosed herein. Diffusetrickling patterns are known in the art (see, e.g., Schmitz-Valckenberget al. Survey of Ophthalmology. 2009 January-February; 54(1):96-117;Bindewald et al. British Journal of Ophthalmology. 2005 July;89(7):874-8; Holz et al. Am. J Ophthalmology. 2007 March; 143(3):463-72,the contents of which are hereby incorporated by reference in theirentireties).

In some embodiments, the dry AMD is identifiable by an increase (e.g. atransient increase) in permeability across the subject's epithelialbarrier between a choroid and a retina, relative to an undiseased state.This is distinct from vascular (endothelial) permeability. For example,this may be seen using angiography.

In some embodiment, RPE toxicity, RPE loss, and the dry AMD areidentifiable using DNIRA, e.g. with sodium iodate (NaIO₃). Areasanalogous to geographic atrophy can be detected by, for example, tissueanalysis (for example, by observing the loss of an RPE cell marker suchas RPE65 and/or the loss of binucleate cell staining), and/or in vivo,using DNIRA, of NaIO₃-treated-eyes. In some embodiments, RPE toxicity,RPE loss and the dry AMD are identifiable using FAF which shows ahyperfluorescent FAF signal, which may be complex, within the area ofimminent tissue loss and/or at its margins. This complexhyperfluorescent FAF signal develops, in the days or weeks after NaIO₃treatment, into a pattern of FAF, which can be complex and can include,but is not limited to ribbon-like, curvilinear, pisciform, scalloped,interlacing, branching, or reticular hyperfluorescence. Such patternsmimic those found in clinical dry AMD. Such FAF may also behypofluorescent.

Further, using angiography alone, or as part of DNIRA, angiographyperformed immediately prior to, or coincident with, the emergence of theareas of altered FAF may demonstrate an increase (including a transientincrease) in permeability across the epithelial barrier between choroidand retina (such as, for example, observed by leakage of dyes that maybe injected prior to imaging; such dyes include ICG). This barriernormally includes the inner choroid, Bruch's Membrane, the RPE cells,and, possibly, the configuration of the outer photoreceptors inconjunction with the RPE and the outer limiting membrane. Withoutwishing to be bound by theory, a transient breakdown of this specializedepithelial barrier may underlie a sequence of events thereafterincluding folding, undulation, or deformation of the outer retina,photoreceptor layer, and/or movement/migration of inflammatory cellsfrom the choroid to the subretinal space. However, in some embodiments,this phase may be transient and resolved, or subclinical in its extent.

In some embodiments, the dry AMD is identifiable by a presence (e.g. aninflux) of immune (e.g. phagocytic immune cells or innate immune cells)cells across the subject's retinal pigment epithelium (RPE) relative toan undiseased state. When there is movement/migration of inflammatorycells from the choroid to the subretinal space, or inner retina to outerretina or subretinal space, such cells may be identified in enucleatedeyes or excised tissue by staining methods known in the art. In theNaIO₃ model, Iba1 staining may be used to detect activated cells of theimmune system. Further, the presence of these cells may be confirmed bycomparison with, for example, NaIO₃ preparations in whichmonocyte/macrophage depletion has been undertaken. Such depletion isknown in the art and may be achieved by treatment with, for example,gadolinium chloride (GAD) or clodronate. In some embodiments, thepresence (e.g. an influx) of immune cells is measured and/or determined,in vivo, by use of DNIRA. Prior to the present invention, such in vivovisualization was not possibly in the clinical setting.

Further, macrophages, an example of an immune cell that may beidentified in the cells of a subject, may be classified by subsets:classically (M1) or alternatively (M2) activated macrophages (see, e.g.,Laskin, Chem Res Toxicol. 2009 Aug. 17; 22(8): 1376-1385, the contentsof which are hereby incorporated by reference in their entireties).Without wishing to be bound by theory, M1 macrophages are activated bystandard mechanisms, such as IFNγ, LPS, and TNFα, while M2 macrophagesare activated by alternative mechanisms, such as IL-4, IL-13, IL-10, andTGFβ. Without wishing to be bound by theory, M1 macrophages display acytotoxic, proinflammatory phenotype, while M2 macrophages, suppresssome aspects of immune and inflammatory responses and participate inwound repair and angiogenesis. In some embodiments, the inventioncomprises the inhibition, modulation or polarization of one or more ofM1 and M2 macrophages.

In some embodiments, the dry AMD is identifiable by the changes in oneor more of blue spectrum fundus imaging, white-light fundus imaging,red-free fundus imaging, and OCT in an eye of the subject. In someembodiments, the dry AMD is identifiable by the changes in one or moreof cSLO, FAF, OCT (including with cross-sectional, three-dimensional anden face viewing), SD-OCT (with cross-sectional, three-dimensional and enface viewing), or other imaging modalities including other wavelengthsof fluorescence (e.g. wavelengths ranging from blue to infrared, e.g.,390 nm to 1 mm, including, for example, blue light, white light,red-free, near infra-red, or infrared). Illustrative changes includedifferences in an imaging pattern in the same of different subjectand/or animal at two different time points and/or experimental orclinical conditions. For example, changes can encompass changes inimaging data between two evaluations of the same subject and/or animaland/or changes in imaging data between a first subject and/or animal andthe imaging data of a second subject and/or animal.

In some embodiments, the AMD is identifiable by OCT. In someembodiments, cross-sectional images show the presence of shallow mounds,or pyramidal or spike-like signals, in the subretinal space.Three-dimensional or enface OCT imaging reveals ribbon-like orcurvilinear, oval, circular, halo or target-like signals. In someembodiments, cross-sectional patterns or transverse patterns areobserved and the cross-sectional or transverse patterns comprise RPEand/or outer retinal loss (atrophy) or mounds, triangles, peaks orspikes found in the sub-retinal space. In various embodiments, thesefeatures are indicative of AMD.

The invention further provides methods for treatment or prevention ofRPD disease, also known as, but not limited to, the following:“reticular drusenoid disease,” “pseudoreticular drusen,” and “drusenoidmacular disease,” or “disease characterized by the presence ofsubretinal drusenoid deposits.”

In some embodiments, the RPD disease is that in which pseudodrusenmaterial is reduced or eradicated or the accumulation and/or expansionof which is slowed upon treatment. In some embodiments, the inventionprovides a method for treating RPD disease in which the pseudodrusen arereduced or eradicated upon treatment in the foveal area and/orperifoveal area and/or juxtafoveal area of a subject's eye.

In some embodiments, the invention further provides methods fortreatment or prevention of RPD disease in which the rates of progressionfrom early to late disease are reduced. Late disease can include, forexample, choroidal neovascularization or geographic atrophy

In some embodiments, the invention further provides methods fortreatment or prevention of RPD disease in which the rates of expansionof geographic atrophy are reduced.

In some embodiments, the RPD disease is identifiable by FAF or otherimaging modalities including other wavelengths of fluorescence. In someembodiments, these wavelengths range from blue to infrared, e.g., 390 nmto 1 mm, including, for example, blue light, white light, near-infrared,and infra-red.

In some embodiments, the RPD disease is identifiable by the presence ofareas of hyper- and/or abnormal-fluorescent FAF or other imagingmodalities including other wavelengths of fluorescence. In someembodiments, these wavelengths range from blue to infrared e.g., 390 nmto 1 mm, including, for example, blue light, white light, near-infraredand infra-red. In some embodiments, the RPD disease is identifiable byusing DNIRA.

In some embodiments, the RPD disease is identifiable using at least oneof, or at least two of, or at least three of, or at least four of whitelight, blue light, FAF, and near infrared and infrared. In a specificembodiment, the RPD disease is identifiable by blue light.

In some embodiments, the RPD disease is identifiable by OCT. In someembodiments, cross-sectional images show the presence of shallow mounds,or pyramidal or spike-like signals, in the subretinal space.Three-dimensional or en face OCT imaging reveals ribbon-like orcurvilinear, oval, circular, halo or target-like signals.

In some embodiments, the RPD is identifiable by an increase (e.g. atransient increase) in permeability across the subject's epithelialbarrier between a choroid and a retina, relative to an undiseased state.This is distinct from vascular (endothelial) permeability. For example,using DNIRA, which comprises, for example, a sodium iodate (NaIO₃) modelof RPE toxicity, geographic areas of RPE damage or loss can be formedthat can be detected by, for example, tissue analysis (for example, byobserving the loss of an RPE cell marker such as RPE65 and/or the lossof binucleate cell staining). Further FAF imaging of NaIO₃-treated RPEshows a hyperfluorescent FAF signal, which may be complex, within thearea of imminent tissue loss and/or at its margins. Thishyperfluorescent FAF signal develops, in the days or weeks after NaIO₃treatment, into a pattern, which can be complex, of FAF that includes,but is not limited to ribbon-like, curvilinear, pisciform, scalloped,interlacing, branching, or reticular hyperfluorescence. Such patternsmimic those found in clinical RPD.

Further, in DNIRA, angiography performed immediately prior to, orcoincident with, the emergence of the areas of altered FAF demonstratesan increase (including a transient increase) in permeability across theepithelial barrier between choroid and retina (such as observed byleakage of dyes that may be injected prior to imaging; such dyes includeICG). This barrier normally includes the inner choroid, Bruch'sMembrane, the RPE cells, and, possibly, the configuration of the outerphotoreceptors in conjunction with the RPE and the outer limitingmembrane. Without wishing to be bound by theory, a transient breakdownof this specialized epithelial barrier may be permissive for thesequence of events thereafter including folding, undulation, ordeformation of the photoreceptor layer. However, in some embodiments,this phase may be transient and resolved, or subclinical in its extent.

In some embodiments, the RPD is identifiable by a presence (e.g. aninflux) of immune cells (e.g. phagocytic immune cells) across thesubject's retinal pigment epithelium (RPE) relative to an undiseasedstate. In some embodiments, presence (e.g. an influx) of immune cells isdetected and/or measured with DNIRA. When there is movement/migration ofinflammatory cells from the choroid to the subretinal space, or innerretina to outer retina or subretinal space, such cells may be identifiedby staining, as is known in the art. For example, using a NaIO₃ model,Iba1 staining may be used to detect activated phagocytic cells of theimmune system. Further, the presence of these cells may be confirmed bycomparison with NaIO₃ preparations in which monocyte/macrophagedepletion has been undertaken. Such depletion is known in the art andmay be achieved by treatment with, for example, gadolinium chloride(GAD) or clodronate. Further, macrophages, an example of a phagocyticimmune cell, that are identified in the cells of a subject, may beclassified by subsets: classically (M1) or alternatively (M2) activatedmacrophages (see, e.g., Laskin, Chem Res Toxicol. 2009 Aug. 17; 22(8):1376-1385, the contents of which are hereby incorporated by reference intheir entireties). Without wishing to be bound by theory, M1 macrophagesare activated by standard mechanisms, such as IFNγ, LPS, and TNFα, whileM2 macrophages are activated by alternative mechanisms, such as IL-4,IL-13, IL-10, and TGFβ. Without wishing to be bound by theory, M1macrophages display a cytotoxic, proinflammatory phenotype, while M2macrophages, suppress some aspects of immune and inflammatory responsesand participate in wound repair and angiogenesis. For example, thepresent invention, in some embodiments, comprises the inhibition,modulation or polarization of one or more of M1 and M2 macrophages.

In some embodiments, the RPD disease is identifiable by the changes inone or more of blue spectrum fundus imaging, white-light fundus imaging,red-free fundus imaging, and OCT in an eye of the subject. In someembodiments, the RPD disease is identifiable by the changes in one ormore of cSLO, FAF, OCT (including with cross-sectional,three-dimensional and en face viewing), SD-OCT (with cross-sectional,three-dimensional and en face viewing), or other imaging modalitiesincluding other wavelengths of fluorescence (e.g. wavelengths rangingfrom blue to infrared, e.g., 390 nm to 1 mm, including, for example,blue light, white light, red-free, near infra-red, or infrared).Illustrative changes include differences in an imaging pattern in thesame of different subject and/or animal at two different time pointsand/or experimental or clinical conditions. For example, changes canencompass changes in imaging data between two evaluations of the samesubject and/or animal and/or changes in imaging data between a firstsubject and/or animal and the imaging data of a second subject and/oranimal.

In another embodiment, the blinding eye disease is LORDs (late onsetretinal degeneration) or retinal degeneration associated with c1qTNF5deficiency or its corresponding gene mutation, or another maculopathy,including, but not limited to, Stargart disease, pattern dystrophy, aswell as retinitis pigmentosa (RP) and related diseases. In oneembodiment, the maculopathy is inherited.

In other embodiments, the blinding eye disease is an idiopathic disorderthat may, without wishing to be bound by theory, be characterized byretinal inflammation, with or without accompanying macular degeneration,including, but not limited to, white-dot syndromes (e.g. serpiginouschorioretinopathy, serpiginous retinopathy, acute posterior multifocalplacoid pigment epitheliopathy (APMPPE), multiple evanescent white dotsyndrome (MEWDS), acute zonal occult outer retinopathy (AZOOR), punctateinner choroidopathy (PIC), and diffuse subretinal fibrosis (DSF)).

In other embodiments, the blinding eye disease is central serousretinopathy (CSR). CSR is a fluid detachment of macula layers from theirsupporting tissue. CSR is often characterizable by the leak andaccumulation of fluid into the subretinal or sub-RPE space. Withoutwishing to be bound by theory, the leak and accumulation of fluid mayoccur because of small breaks in the RPE.

In some embodiments, one or more of the blinding eye diseases areidentifiable by the presence of areas of hyper- and/orabnormal-fluorescent FAF, or other wavelengths of light from 350 nm to1,000 nm. In some embodiments, one or more of the blinding eye diseasesare identifiable using DNIRA.

In some embodiments, one or more of the blinding eye diseases areidentifiable by, for example, cSLO, FAF, OCT (including withcross-sectional, three-dimensional and en face viewing), SD-OCT (withcross-sectional, three-dimensional and en face viewing), or otherimaging modalities including other wavelengths of fluorescence (e.g.wavelengths ranging from blue to infrared, e.g., 390 nm to 1 mm,including, for example, blue light, white light, red-free, nearinfra-red, or infrared).

In some embodiments, the blinding eye disease may be of a certain stageor progression. In some embodiments, the blinding eye disease may beincipient, emerging, quiescent, advancing or active.

In addition to treating pre-existing blinding eye diseases, the presentinvention comprises prophylactic methods in order to prevent or slow theonset of these disorders. In prophylactic applications, an agent can beadministered to a subject susceptible to or otherwise at risk of aparticular blinding eye disease. Such susceptibility may be determinedby, for example, familial predisposition, genetic testing, risk factoranalysis, blood or other cytokine or biomarker levels, and ocularexamination, which can include multi-modal analysis such as FAF, bluelight, white light, red-free, near infra-red, infrared, DNIRA, etc. Suchsusceptibility may also be determined by, for example, detection by OCT,with cross-sectional, three-dimensional and enface viewing.

In addition to treating defined, known blinding eye diseases, thepresent invention comprises particular patterns of in vivo imaging usinglight at wavelengths ranging from 300 to 1,000 nm, including whitelight, blue light, FAF, infra-red, near infra-red, DNIRA or by OCT, withcross-sectional, three-dimensional and enface viewing. In applicationsagainst particular patterns of in vivo imaging, an agent can beadministered to a subject with, susceptible to, or otherwise at risk ofa particular blinding disease. Such diagnosis or susceptibility may bedetermined by, for example, ophthalmic examination, familialpredisposition, genetic testing, risk factor analysis, and blood orother cytokine or biomarker levels, which can include multi-modalanalysis such as FAF, blue light, white light, red-free, near infra-red,infrared, DNIRA, etc. Such susceptibility may also be determined by, forexample, detection by OCT, with cross-sectional, three-dimensional anden face viewing.

Agents of the Invention

In various aspects, the present invention provides for theidentification and use of a candidate compound and/or test compound. Inembodiments providing for identification and use of a candidate compoundand/or test compound, the candidate compound and/or test compound may bechemical, molecule, compound, biologic (e.g. an antibody or peptide),drug, pro-drug, cellular therapy, low molecular weight syntheticcompound, or a small molecule drug. In some embodiments, the candidatecompound and/or test compound is selected from a library of compoundsknown in the art. In some embodiments, the candidate compound is usefulfor treating a blinding eye disease, preventing a binding eye disease,or reducing the rate of pathogenesis of a blinding eye disease.

In various aspects, the present invention provides for a method fortreating or preventing dry AMD and/or RPD. In some embodiments, acompound of Formula I, methotrexate, or a pharmaceutically acceptablesalt thereof, is also administered with an additional therapeutic agent,including, for example, one or more of an anti-VEGF agent, an ACEinhibitor, a PPAR-gamma agonist or partial agonist, a renin inhibitor, asteroid, and an agent that modulates autophagy, as well as a semapimod,a MIF inhibitor, a CCR2 inhibitor, CKR-2B, a 2-thioimidazole, CAS445479-97-0, CCX140, clodronate, a clodonate-liposome preparation orgadolinium chloride.

In various aspects, the present invention provides for identifying ablinding eye disease subject who is more likely than not to respond totreatment with an agent (an “agent of the invention”) and/or determiningwhether a blinding eye disease in a subject is responsive to treatmentwith an agent.

In some embodiments, an agent of the invention is a compound of FormulaI:

or a pharmaceutically acceptable salts thereof, wherein each of R₁ andR₂ is independently H or a C₁-C₆ alkyl and R₃ is H or a C₁-C₆ alkyl.

In various embodiments, R₁ and R₂ are independently hydrogen, methyl orethyl.

In some embodiments R₁ and R₂ are independently methyl or ethyl.

In some embodiments R₁ and R₂ are methyl.

In some embodiments R₃ is hydrogen, methyl or ethyl.

In some embodiments R₃ is hydrogen.

In some embodiments, particularly where R₃ is hydrogen, the compounds ofFormula I are in the form of a pharmaceutically acceptable salt.

In some embodiments R₁ and R₂ are methyl and R₃ is hydrogen. In suchembodiments, the compound of Formula I is 2-((1-benzylindazol-3-yl)methoxy)-2-methyl propionic acid, also known as2-methyl-2-[[1-(phenylmethyl)-1H-indazol-3yl]methoxy] propanoic acid.Such a compound is commonly known as bindarit and has the followingstructure:

Synthesis of bindarit is described in detail in U.S. Pat. No. 4,999,367,which is hereby incorporated by reference in its entirety.

Bindarit is an inhibitor of MCP-1 production in vitro and in vivo and,without wishing to be bound by theory, its beneficial effects in animalmodels of inflammation may be related to this anti-MCP-1 activity (seeMirolo, et al. Eur Cytokine Netw 2008; 19:119-122). Bindarit has alsobeen shown to selectively inhibit the production of MCP-2 and MCP-3 (seeMirolo, et al. Eur Cytokine Netw 2008; 19:119-122).

In some embodiments, an agent of the invention is methotrexate or apharmaceutically acceptable salt thereof. Methotrexate has thestructure:

Methotrexate is also known by its IUPAC name,(2S)-2-[(4-{[(2,4-diaminopteridin-6-yl)methyl](methyl)amino}benzoyl)amino]pentanedioicacid; by “MTX” and by “amethopterin.” Methotrexate sodium is availableby prescription in the United States. Methotrexate's synthesis has beendescribed in U.S. Pat. No. 4,080,325, which is hereby incorporated byreference in its entirety.

In still another embodiment, an agent of the invention is a modulator ofmacrophage polarization. Illustrative modulators of macrophagepolarization include peroxisome proliferator activated receptor gamma(PPAR-g) modulators, including, for example, agonists, partial agonists,antagonists or combined PPAR-gamma/alpha agonists.

In some embodiments, the PPAR gamma modulator is a full agonist or apartial agonist. In some embodiments, the PPAR gamma modulator is amember of the drug class of thiazolidinediones (TZDs, or glitazones). Byway of non-limiting example, the PPAR gamma modulator may be one or moreof rosiglitazone (AVANDIA), pioglitazone (ACTOS), troglitazone(REZULIN), netoglitazone, rivoglitazone, ciglitazone, rhodanine. In someembodiments, the PPAR gamma modulator is one or more of irbesartan andtelmesartan. In some embodiments, the PPAR gamma modulator is anonsteroidal anti-inflammatory drugs (NSAID, such as, for example,ibuprofen) and indoles. Known inhibitors include the experimental agentGW-9662. Further examples of PPAR gamma modulators are described in WIPOPublication Nos. WO/1999/063983, WO/2001/000579, Nat Rev Immunol. 2011Oct. 25; 11(11):750-61, or agents identified using the methods ofWO/2002/068386, the contents of which are hereby incorporated byreference in their entireties.

In some embodiments, the PPAR gamma modulator is a “dual,” or“balanced,” or “pan” PPAR modulator. In some embodiments, the PPAR gammamodulator is a glitazar, which bind two or more PPAR isoforms, e.g.,muraglitazar (Pargluva) and tesaglitazar (Galida) and aleglitazar.

In another embodiment, an agent of the invention is semapimod (CNI-1493)as described in Bianchi, et al. (March 1995). Molecular Medicine(Cambridge, Mass.) 1 (3): 254-266, the contents of which are herebyincorporated by reference in their entireties.

In still another embodiment, an agent of the invention is a migrationinhibitory factor (MIF) inhibitor. Illustrative MIF inhibitors aredescribed in WIPO Publication Nos. WO 2003/104203, WO 2007/070961, WO2009/117706 and U.S. Pat. Nos. 7,732,146 and 7,632,505, and 7,294,7537,294,753 the contents of which are hereby incorporated by reference intheir entireties. In some embodiments, the MIF inhibitor is(S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methylester (ISO-1), isoxazoline, p425 (J. Biol. Chem., 287, 30653-30663),epoxyazadiradione, or vitamin E.

In still another embodiment, an agent of the invention is a chemokinereceptor 2 (CCR2) inhibitor as described in, for example, U.S. patentand patent Publication Nos.: U.S. Pat. Nos. 7,799,824, 8,067,415, US2007/0197590, US 2006/0069123, US 2006/0058289, and US 2007/0037794, thecontents of which are hereby incorporated by reference in theirentireties. In some embodiments, the CCR2) inhibitor is Maraviroc,cenicriviroc, CD192, CCX872, CCX140,2-((Isopropylaminocarbonyl)amino)-N-(2-((cis-2-((4-(methylthio)benzoyl)amino)cyclohexyl)amino)-2-oxoethyl)-5-(trifluoromethyl)-benzamide,vicriviroc, SCH351125, TAK779, Teijin, RS-504393, compound 2, compound14, or compound 19 (Plos ONE 7(3): e32864).

In various specific embodiments, an agent of the invention is one ormore of CKR-2B, a 2-thioimidazole, CCR2 Antagonist (CAS 445479-97-0),and CCX140.

In yet another embodiment, an agent of the invention is a clodronate. Instill another embodiment, the agent is a clodronate liposome preparationas described in Barrera et al. Arthritis and Rheumatism, 2000, 43, pp.1951-1959, the contents of which are hereby incorporated by reference intheir entireties.

In still another embodiment, an agent of the invention is a chelated orunchelated form of gadolinium, for example gadolinium chloride (GAD).

In various embodiments an agent of the invention is an anti-VEGF agent.Non limiting examples of anti-VEGF agents useful in the present methodsinclude ranibizumab, bevacizumab, aflibercept, KH902 VEGF receptor-Fc,fusion protein, 2C3 antibody, ORA102, pegaptanib, bevasiranib,SIRNA-027, decursin, decursinol, picropodophyllin, guggulsterone,PLG101, eicosanoid LXA4, PTK787, pazopanib, axitinib, CDDO-Me, CDDO-Imm,shikonin, beta-, hydroxyisovalerylshikonin, ganglioside GM3, DC101antibody, Mab25 antibody, Mab73 antibody, 4A5 antibody, 4E10 antibody,5F12 antibody, VA01 antibody, BL2 antibody, VEGF-related protein,sFLT01, sFLT02, Peptide B3, TG100801, sorafenib, G6-31 antibody, afusion antibody and an antibody that binds to an epitope of VEGF.Additional non-limiting examples of anti-VEGF agents useful in thepresent methods include a substance that specifically binds to one ormore of a human vascular endothelial growth factor-A (VEGF-A), humanvascular endothelial growth factor-B (VEGF-B), human vascularendothelial growth factor-C (VEGF-C), human vascular endothelial growthfactor-D (VEGF-D) and human vascular endothelial growth, factor-E(VEGF-E), and an antibody that binds, to an epitope of VEGF.

In one embodiment, the anti-VEGF agent is the antibody ranibizumab or apharmaceutically acceptable salt thereof. Ranibizumab is commerciallyavailable under the trademark LUCENTIS. In another embodiment, theanti-VEGF agent is the antibody bevacizumab or a pharmaceuticallyacceptable salt thereof. Bevacizumab is commercially available under thetrademark AVASTIN. In another embodiment, the anti-VEGF agent isaflibercept or a pharmaceutically acceptable salt thereof. Afliberceptis commercially available under the trademark EYLEA. In one embodiment,the anti-VEGF agent is pegaptanib or a pharmaceutically acceptable saltthereof. Pegaptanib is commercially available under the trademarkMACUGEN. In another embodiment, the anti-VEGF agent is an antibody or anantibody fragment that binds to an epitope of VEGF, such as an epitopeof VEGF-A, VEGF-B, VEGF-C, VEGF-D, or VEGF-E. In some embodiments, theVEGF antagonist binds to an epitope of VEGF such that binding of VEGFand VEGFR are inhibited. In one embodiment, the epitope encompasses acomponent of the three dimensional structure of VEGF that is displayed,such that the epitope is exposed on the surface of the folded VEGFmolecule. In one embodiment, the epitope is a linear amino acid sequencefrom VEGF.

In various embodiments, an agent of the invention is a renin angiotensinsystem (RAS) inhibitor. In some embodiments, the renin angiotensinsystem (RAS) inhibitor is one or more of an angiotensin-convertingenzyme (ACE) inhibitor, an angiotensin-receptor blocker, and a renininhibitor.

Non limiting examples of angiotensin-converting enzyme (ACE) inhibitorswhich are useful in the present invention include, but are not limitedto: alacepril, alatriopril, altiopril calcium, ancovenin, benazepril,benazepril hydrochloride, benazeprilat, benzazepril, benzoylcaptopril,captopril, captoprilcysteine, captoprilglutathione, ceranapril,ceranopril, ceronapril, cilazapril, cilazaprilat, converstatin,delapril, delaprildiacid, enalapril, enalaprilat, enalkiren, enapril,epicaptopril, foroxymithine, fosfenopril, fosenopril, fosenopril sodium,fosinopril, fosinopril sodium, fosinoprilat, fosinoprilic acid,glycopril, hemorphin-4, idapril, imidapril, indolapril, indolaprilat,libenzapril, lisinopril, lyciumin A, lyciumin B, mixanpril, moexipril,moexiprilat, moveltipril, muracein A, muracein B, muracein C, pentopril,perindopril, perindoprilat, pivalopril, pivopril, quinapril, quinaprilhydrochloride, quinaprilat, ramipril, ramiprilat, spirapril, spiraprilhydrochloride, spiraprilat, spiropril, spirapril hydrochloride,temocapril, temocapril hydrochloride, teprotide, trandolapril,trandolaprilat, utibapril, zabicipril, zabiciprilat, zofenopril,zofenoprilat, pharmaceutically acceptable salts thereof, and mixturesthereof.

Non limiting examples of angiotensin-receptor blockers which are usefulin the present invention include, but are not limited to: irbesartan(U.S. Pat. No. 5,270,317, hereby incorporated by reference in itsentirety), candesartan (U.S. Pat. Nos. 5,196,444 and 5,705,517 herebyincorporated by reference in their entirety), valsartan (U.S. Pat. No.5,399,578, hereby incorporated by reference in its entirety), andlosartan (U.S. Pat. No. 5,138,069, hereby incorporated by reference inits entirety).

Non limiting examples of renin inhibitors which are useful in thepresent invention include, but are not limited to: aliskiren, ditekiren,enalkiren, remikiren, terlakiren, ciprokiren and zankiren,pharmaceutically acceptable salts thereof, and mixtures thereof.

In various embodiments an agent of the invention is a steroid. In someembodiments, a steroid is a compound belonging to or related to thefollowing illustrative families of compounds: corticosteroids,mineralicosteroids, and sex steroids (including, for example,potentially androgenic or estrogenic or anti-androgenic andanti-estrogenic molecules). Included amongst these are, by way ofnon-limiting example, prednisone, prednisolone, methyl-prednisolone,triamcinolone, fluocinolone, aldosterone, spironolactone, danazol(otherwise known as OPTINA), and others.

In various embodiments an agent of the invention is an agent thatmodulates autophagy, microautophagy, mitophagy or other forms ofautophagy. In some embodiments, the candidate drug and/or compound isone or more of sirolimus, tacrolimis, rapamycin, everolimus,bafilomycin, chloroquine, hydroxychloroquine, spautin-1, metformin,perifosine, resveratrol, trichostatin, valproic acide, Z-VAD-FMK, orothers known to those in the art. Without wishing to be bound by theory,agent that modulates autophagy, microautophagy, mitophagy or other formsof autophagy may alter the recycling of intra-cellular components, forexample, but not limited to, cellular organelles, mitochondria,endoplasmic reticulum, lipid or others. Without further wishing to bebound by theory, this agent may or may not act throughmicrotubule-associated protein 1A/1B-light chain 3 (LC3).

Fluorescent Compounds

In some embodiments, the fluorescent compound is suitable for imagingwith various wavelengths of fluorescence. In some embodiments, thesewavelengths range from visible light to infrared, e.g., 390 nm to 1 mm,including, for example, blue light, white light, and near-infrared. Insome embodiments, the dye is a near-infrared dye. In some embodiments,the dye is ICG.

In some embodiments, the fluorescent compound is suitable for imagingwith various wavelengths of fluorescence. In some embodiments, thesewavelengths range from visible light to infrared, e.g., about 390 nm toabout 1 mm, including, for example, blue light, white light, andnear-infrared. In some embodiments, the absorption is from about 390 nmto about 1 mm. In some embodiments, the emission is from about 390 nm toabout 1 mm.

In some embodiments, the fluorescent compound absorbs light at awavelength of about 600 nm to about 900 nm and/or emits light at awavelength of about 750 nm to about 950 nm.

In some embodiments, the dye is a near-infrared dye. In someembodiments, the dye is ICG. In some embodiments, the ICGexcitation/emission filters are 795 nm (excitation)/810 nm (emission).

In some embodiments, the dose of the fluorescent compound, e.g. a dye(including ICG), is an effective amount of the fluorescent compound. Invarious embodiments, the dose of ICG is from about 0.1 to about 10 mg/kgof an animal. In some embodiments, the dose is about 0.1, or about 0.3,or about 0.5, or about 1.0, or about 2.0, or about 3.0, or about 4.0, orabout 5.0, or about 6.0, or about 7.0, or about 8.0, or about 9.0, orabout 10.0 mg/kg of an animal.

In various embodiments, the fluorescent compounds, or metabolitesthereof, cause a fluorescence which occurs in RPE cells and/or immunecells.

In other embodiments, the methods described herein do not compriseadministering (i) an additional amount of fluorescent compound to theanimal or (ii) a second fluorescent compound to the animal.

Toxins

In some embodiments, a toxin known to affect ocular tissue, includingbut not limited to, the RPE or retina, is provided.

In some embodiments, the toxin is one or more of aluminium,aminophenoxyalkanes (a non-limiting example includes, but is not limitedto, 1,4,-bis(4-aminophenoxy)-2-phenylbenzene to rats), cationicamphophilic drugs/tricyclic antidepressants (non-limiting examplesinclude but are not limited to amiodarone, chloroamitriptyline,chlorphentermine, clomipramine, imipramine, iprindole, variousaminoglycosides, and other cationic amphophilic compounds),desferrioxamine, dl-(p-trifluoromethylphenyl) isopropylaminehydrochloride, fluoride (e.g. sodium fluoride), iodate (non-limitingexamples include but are not limited to, sodium or potassium iodate),iodoacetate, lead, methanol and formic acid, 4,4′-Methylenedianiline,N-methyl-N-nitrosurea, naphthalene, naphthol, nitroaniline(N-3-pyridylmethyl-N′-p-nitrophenylurea/nitroanilin/pyriminil),organophosphates (non-limiting examples include but are not limited toethylthiometon, fenthion, and fenitrothion), oxalate (a non-limitingexample includes but are not limited to dibutyl oxalate), phenothiazines(non-limiting examples include but are not limited topiperidylchlorophenothiazine, thioridazine, and chlorpromazine),quinolines (a non-limiting example includes, but is not limited to,chloroquine and hydroxychloroquine), streptozotocin, taurine deficiency,urethane, zinc deficiency caused by metal chelators, and derivatives andvariants thereof.

In some embodiments, the toxin is an iodate. In some embodiments, thetoxin is sodium or potassium iodate.

In some embodiments, the toxin induces atrophy of ocular tissue. Invarious embodiments, the atrophy comprises necrosis and/or apoptosis. Invarious embodiments, the atrophy comprising necrosis and/or apoptosis isof RPE cells. In some embodiments, the toxin reduces or modifiesautophagy. In some embodiments, the toxin induces geographic atrophyand/or the expansion of geographic atrophy. In some embodiments, thetoxin induces one or more of the above-mentioned effects.

In some embodiments, the toxin is administered one time, or two times,or three times. In some embodiments, the toxin is administered one time,or two times, or three times, or four times, or five times. In someembodiments, a second, or third, or fourth, or fifth administration iswithin about a day, or 1 week, or 1 month of the first.

In some embodiments, the toxin administered may be may be one or more ofthe agents described herein. In various embodiments, the second, orthird, or fourth, or fifth pulse is a modulator of autophagy, cellsurvival, cell death, proliferation, regeneration, and the like, asdescribed herein and as known in the art.

In another embodiment, the methods described herein compriseadministering (i) an additional amount of a first toxin to the animaland/or (ii) a second toxin to the animal. In some embodiments, themethods described herein comprise further comprise observing a reductionin the rate of formation, growth or expansion of patches of oculartissue atrophy or patches of tissue loss.

In various embodiments, doses of the toxins are known to those in theart. For example, a suitable dosage may be in a range of about 0.1 mg/kgto about 100 mg/kg of body weight of the subject, for example, about 0.1mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9mg/kg, about 1 mg/kg, about 1.1 mg/kg, about 1.2 mg/kg, about 1.3 mg/kg,about 1.4 mg/kg, about 1.5 mg/kg, about 1.6 mg/kg, about 1.7 mg/kg,about 1.8 mg/kg, about 1.9 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about9 mg/kg, about 10 mg/kg, about 11 mg/kg, about 12 mg/kg, about 13 mg/kg,about 14 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg,about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg,or about 100 mg/kg body weight, inclusive of all values and rangestherebetween.

In one embodiment, the toxin is NaIO₃ and the dose is about 50 mg/kg ofbody weight. In one embodiment, the toxin is NaIO₃ and the dose is about45 mg/kg of body weight. In one embodiment, the toxin is NaIO₃ and thedose is about 30 mg/kg of body weight.

Pharmaceutically Acceptable Salts and Excipients

Any agent described herein can possess a sufficiently basic functionalgroup, which can react with an inorganic or organic acid, or a carboxylgroup, which can react with an inorganic or organic base, to form apharmaceutically acceptable salt. A pharmaceutically acceptable acidaddition salt is formed from a pharmaceutically acceptable acid, as iswell known in the art. Such salts include the pharmaceuticallyacceptable salts listed in Journal of Pharmaceutical Science, 66, 2-19(1977) and The Handbook of Pharmaceutical Salts; Properties, Selection,and Use. P. H. Stahl and C. G. Wermuth (eds.), Verlag, Zurich(Switzerland) 2002, which are hereby incorporated by reference in theirentirety.

Pharmaceutically acceptable salts include, by way of non-limitingexample, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide,nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate,camphorsulfonate, pamoate, phenylacetate, trifluoroacetate, acrylate,chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate,methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, isobutyrate,phenylbutyrate, α-hydroxybutyrate, butyne-1,4-dicarboxylate,hexyne-1,4-dicarboxylate, caprate, caprylate, cinnamate, glycollate,heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate,mesylate, nicotinate, phthalate, teraphthalate, propiolate, propionate,phenylpropionate, sebacate, suberate, p-bromobenzenesulfonate,chlorobenzenesulfonate, ethylsulfonate, 2-hydroxyethylsulfonate,methylsulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,naphthalene-1,5-sulfonate, xylenesulfonate, and tartarate salts.

The term “pharmaceutically acceptable salt” also refers to a salt of thecompounds of the present invention having an acidic functional group,such as a carboxylic acid functional group, and a base. Suitable basesinclude, but are not limited to, hydroxides of alkali metals such assodium, potassium, and lithium; hydroxides of alkaline earth metal suchas calcium and magnesium; hydroxides of other metals, such as aluminumand zinc; ammonia, and organic amines, such as unsubstituted orhydroxy-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine;tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine;triethylamine; mono-, bis-, or tris-(2-OH-lower alkylamines), such asmono-; bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine,or tris-(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxyl-loweralkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine ortri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such asarginine, lysine, and the like.

In some embodiments, an agent of the invention is in the form or apharmaceutically acceptable salt. In some embodiments, thepharmaceutically acceptable salt is a sodium salt. In some embodiments,particularly where R₃ of the compounds of Formula I is hydrogen, thecompounds of Formula I are in the form of a pharmaceutically acceptablesalt. In some embodiments, the compound of Formula I is apharmaceutically acceptable salt of bindarit. In some embodiments,methotrexate is in the form or a pharmaceutically acceptable salt. Insome embodiments, the pharmaceutically acceptable salt of methotrexateis methotrexate sodium.

Further, any agent described herein can be administered to a subject asa component of a composition that comprises a pharmaceuticallyacceptable carrier or vehicle. Such compositions can optionally comprisea suitable amount of a pharmaceutically acceptable excipient so as toprovide the form for proper administration.

Pharmaceutical excipients can be liquids, such as water and oils,including those of petroleum, animal, vegetable, or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.The pharmaceutical excipients can be saline, gum acacia, gelatin, starchpaste, talc, keratin, colloidal silica, urea and the like. In addition,auxiliary, stabilizing, thickening, lubricating, and coloring agents canbe used. In one embodiment, the pharmaceutically acceptable excipientsare sterile when administered to a subject. Water is a useful excipientwhen any agent described herein is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid excipients, specifically for injectable solutions.Suitable pharmaceutical excipients also include starch, glucose,lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodiumstearate, glycerol monostearate, talc, sodium chloride, dried skim milk,glycerol, propylene, glycol, water, ethanol and the like. Any agentdescribed herein, if desired, can also comprise minor amounts of wettingor emulsifying agents, or pH buffering agents.

Formulations, Administration, and Dosing

Any agent described herein can take the form of solutions, suspensions,emulsion, intra-ocular injection, intra-vitreal injection, topicalophthalmic drops, sub-conjunctival injection, sub-Tenon's injection,trans-scleral formulations, tablets, pills, pellets, capsules, capsulescontaining liquids, powders, sustained-release formulations,suppositories, emulsions, aerosols, sprays, suspensions, or any otherform suitable for use. In one embodiment, the composition is suitablefor an intra-vitreal injection (see, e.g., ILUVIEN or similar forms) orimplantation (see, e.g., RETISERT or similar forms). In one embodiment,the composition is in the form of a capsule (see, e.g., U.S. Pat. No.5,698,155). Other examples of suitable pharmaceutical excipients aredescribed in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso R.Gennaro eds., 19th ed. 1995), incorporated herein by reference.

In one embodiment, any agent described herein is formulated forophthalmic administration, including, for example, intravitreal orintraocular administration, and/or intravenous administration.Typically, compositions for administration comprise sterile isotonicaqueous buffer. Where necessary, the compositions can also include asolubilizing agent. Also, the agents can be delivered with a suitablevehicle or delivery device as known in the art. Combination therapiesoutlined herein can be co-delivered in a single delivery vehicle ordelivery device. Compositions for administration can optionally includea local anesthetic such as lignocaine to lessen pain at the site of theinjection.

In one embodiment, any agent described herein is formulated inaccordance with routine procedures as a composition adapted forintra-ocular administration.

In one embodiment, any agent described herein is formulated inaccordance with routine procedures as a composition adapted for oraladministration to human beings. Compositions for oral delivery can be inthe form of tablets, lozenges, aqueous or oily suspensions, granules,powders, emulsions, capsules, syrups, or elixirs, for example. Orallyadministered compositions can comprise one or more agents, for example,sweetening agents such as fructose, aspartame or saccharin; flavoringagents such as peppermint, oil of wintergreen, or cherry; coloringagents; and preserving agents, to provide a pharmaceutically palatablepreparation. Moreover, where in tablet or pill form, the compositionscan be coated to delay disintegration and absorption in thegastrointestinal tract thereby providing a sustained action over anextended period of time. Selectively permeable membranes surrounding anosmotically active driving any agent described herein are also suitablefor orally administered compositions. In these latter platforms, fluidfrom the environment surrounding the capsule is imbibed by the drivingcompound, which swells to displace the agent or agent compositionthrough an aperture. These delivery platforms can provide an essentiallyzero order delivery profile as opposed to the spiked profiles ofimmediate release formulations. A time-delay material such as glycerolmonostearate or glycerol stearate can also be useful. Oral compositionscan include standard excipients such as mannitol, lactose, starch,magnesium stearate, sodium saccharin, cellulose, and magnesiumcarbonate. In one embodiment, the excipients are of pharmaceuticalgrade.

The ingredients may be supplied either separately or mixed together inunit dosage form, for example, as a pre-mixed solution, drylyophilized-powder, or water-free concentrate in a hermetically sealedcontainer such as an ampule, pre-filled syringe, or sachette indicatingthe quantity of active agent. Where any agent described herein is to beadministered by intra-vitreal or intra-ocular delivery, it can bedispensed, for example, with a pre-filled syringe or injector, or in anampule for withdrawal into a suitable syringe. Where any agent describedherein is to be administered by infusion, it can be dispensed, forexample, with an infusion bottle containing sterile pharmaceutical gradewater or saline. Where any agent described herein is to be administeredby injection, an ampule of sterile water for injection or saline can beprovided so that the ingredients can be mixed prior to administration.

Any agent described herein can be administered by controlled-release orsustained-release means or by delivery devices that are well known tothose of ordinary skill in the art. Examples include, but are notlimited to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899;3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767;5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,556, each of whichis incorporated herein by reference in its entirety. Such dosage formscan be useful for providing controlled- or sustained-release of one ormore active ingredients using, for example, hydropropylmethyl cellulose,other polymer matrices, gels, permeable membranes, osmotic systems,multilayer coatings, microparticles, liposomes, microspheres, or acombination thereof to provide the desired release profile in varyingproportions. Suitable controlled- or sustained-release formulationsknown to those skilled in the art, including those described herein, canbe readily selected for use with the active ingredients of the agentsdescribed herein. The invention thus provides single unit dosage formssuitable for oral administration such as, but not limited to, tablets,capsules, gelcaps, and caplets that are adapted for controlled- orsustained-release.

Controlled- or sustained-release of an active ingredient can bestimulated by various conditions, including but not limited to, changesin pH, changes in temperature, stimulation by an appropriate wavelengthof light, concentration or availability of enzymes, concentration oravailability of water, or other physiological conditions or compounds.

Compositions can be prepared according to conventional mixing,granulating, coating or polymerization methods, respectively, and thepresent compositions can comprise, in one embodiment, from about 0.1% toabout 99%; and in another embodiment from about 1% to about 70% of anyagent described herein by weight or volume.

In another embodiment, any agent described herein acts synergisticallywhen co-administered with another agent and is administered at dosesthat are lower than the doses commonly employed when such agents areused as monotherapy.

For example, the dosage any agent described herein as well as the dosingschedule can depend on various parameters, including, but not limitedto, the blinding eye disease being treated, the subject's generalhealth, and the administering physician's discretion. Any agentdescribed herein, can be administered prior to (e.g., 5 minutes, 15minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks,4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concurrentlywith, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 8 weeks, or 12 weeks after) the administration of an additionaltherapeutic agent, to a subject in need thereof. In various embodimentsany agent described herein is administered 1 minute apart, 10 minutesapart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hoursapart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10hours apart, 10 hours to 11 hours apart, 11 hours to 12 hours apart, nomore than 24 hours apart or no more than 48 hours apart. In oneembodiment, an agent of the invention, including compounds of Formula I,methotrexate or their pharmaceutically acceptable salts, and one or moreadditional therapeutic agents are administered within 3 hours. Inanother embodiment, an agent of the invention, including compounds ofFormula I, methotrexate or their pharmaceutically acceptable salts, andone or more additional therapeutic agents are administered at 1 minuteto 24 hours apart.

The amount of any agent described herein that is admixed with thecarrier materials to produce a single dosage can vary depending upon thesubject being treated and the particular mode of administration. Invitro or in vivo assays can be employed to help identify optimal dosageranges.

In general, the doses that are useful are known to those in the art. Forexample, doses may be determined with reference Physicians' DeskReference, 66th Edition, PDR Network; 2012 Edition (Dec. 27, 2011), thecontents of which are incorporated by reference in its entirety.

The dosage of any agent described herein can depend on several factorsincluding the severity of the condition, whether the condition is to betreated or prevented, and the age, weight, and health of the subject tobe treated. Additionally, pharmacogenomic (the effect of genotype on thepharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic)information about a particular subject may affect dosage used.Furthermore, the exact individual dosages can be adjusted somewhatdepending on a variety of factors, including the specific combination ofthe agents being administered, the time of administration, the route ofadministration, the nature of the formulation, the rate of excretion,the particular blinding eye disease being treated, the severity of thedisorder, and the anatomical location of the disorder. Some variationsin the dosage can be expected.

In some embodiments, the administering is effected orally orintra-vascularly, or intraocularly, or periocularly, or to the ocularsurface

When ophthalmically administered to a human, for example,intravitreally, the dosage of an agent of the invention, including, forexample, Formula I, methotrexate or a pharmaceutically acceptable saltthereof and/or additional therapeutic agent is normally 0.003 mg to 5.0mg per eye per administration, or 0.03 mg to 3.0 mg per eye peradministration, or 0.1 mg to 1.0 mg per eye per administration. In oneembodiment, the dosage is 0.03 mg, 0.3 mg, 1.5 mg or 3.0 mg per eye. Inanother embodiment, the dosage is 0.5 mg per eye. The dosage can rangefrom 0.01 mL to 0.2 mL administered per eye, or 0.03 mL to 0.15 mLadministered per eye, or 0.05 mL to 0.10 mL administered per eye. In oneembodiment, the administration is 400 μg of compound, monthly for atleast three months.

Generally, when orally administered to a mammal, the dosage of any agentdescribed herein may be 0.001 mg/kg/day to 100 mg/kg/day, 0.01 mg/kg/dayto 50 mg/kg/day, or 0.1 mg/kg/day to 10 mg/kg/day. When orallyadministered to a human, the dosage of any agent described herein isnormally 0.001 mg to 1000 mg per day, 1 mg to 600 mg per day, or 5 mg to30 mg per day. In one embodiment, oral dosage is 600 mg per day. In oneembodiment, the oral dosage is two 300 mg doses per day. In anotherembodiment, oral dosage is 7.5 mg per week to 15 mg per week.

For administration of any agent described herein by parenteralinjection, the dosage is normally 0.1 mg to 250 mg per day, 1 mg to 20mg per day, or 3 mg to 5 mg per day. Injections may be given up to fourtimes daily. Generally, when orally or parenterally administered, thedosage of any agent described herein is normally 0.1 mg to 1500 mg perday, or 0.5 mg to 10 mg per day, or 0.5 mg to 5 mg per day. A dosage ofup to 3000 mg per day can be administered.

In some embodiments, it may be desirable to administer one or more anyagent described herein to the eye. Administration may be, by way ofnon-limiting example, intra-ocular, intra-vitreal, topical (including,but not limited to, drops and ointment), sub-conjunctival, sub-Tenon's,trans-scleral, suprachoroidal, subretinal, and via iontophoresis.

Other routes of administration may also be used, such as, for example:intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,intranasal, epidural, oral, sublingual, intranasal, intracerebral,intravaginal, transdermal, rectally, by inhalation, or topically,particularly to the ears, nose, eyes, or skin.

The mode of administration can be left to the discretion of thepractitioner, and depends in-part upon the site of the medicalcondition. In most instances, administration results in the release ofany agent described herein into the bloodstream.

Any agent described herein can be administered orally. Such agents canalso be administered by any other convenient route, for example, byintravenous infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and can be administered together with anotherbiologically active agent. Administration can be systemic or local.Various delivery systems are known, e.g., encapsulation in liposomes,microparticles, microcapsules, capsules, etc., and can be used toadminister.

Further methods of administration include but are not limited tointra-ocular, intra-vitreal, topical ocular (including but not limitedto drops, ointments and inserts), sub-conjunctival, sub-Tenon's,suprachoroidal, trans-scleral, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral,sublingual, intranasal, intracerebral, intravaginal, transdermal,rectally, by inhalation, or topically, particularly to the ears, nose,eyes, or skin. In some embodiments, more than one of any agent describedherein is administered to the eye. Administration may be, by way ofnon-limiting example, intra-ocular, intra-vitreal, topical (including,but not limited to, drops and ointment), sub-conjunctival, sub-Tenon's,trans-scleral, and iontophoresis. The mode of administration can be leftto the discretion of the practitioner, and depends in-part upon the siteof the medical condition. In most instances, administration results inthe release into the bloodstream.

In specific embodiments, it may be desirable to administer locally tothe area in need of treatment.

In another embodiment, delivery can be in a vesicle, in particular aliposome (see Langer, 1990, Science 249:1527-1533; Treat et al., inLiposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989).In yet another embodiment, delivery can be in a controlled releasesystem. In one embodiment, a slow release intra-ocular device may beused. In some embodiments, this device consists of a locally deliverederodible or non-erodible liquid, gel, polymer, etc.

In another embodiment, polymeric materials can be used (see MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Pres.,Boca Raton, Florida (1974); Controlled Drug Bioavailability, DrugProduct Design and Performance, Smolen and Ball (eds.), Wiley, New York(1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem.23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989,Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105). Inanother embodiment, a controlled-release system can be placed inproximity of the target area to be treated, e.g., the retina, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138(1984)). Other controlled-release systems discussed in the review byLanger, 1990, Science 249:1527-1533) may be used.

Administration of any agent described herein can, independently, be oneto four times daily or one to four times per month or one to six timesper year or once every two, three, four or five years. Administrationcan be for the duration of one day or one month, two months, threemonths, six months, one year, two years, three years, and may even befor the life of the subject. Chronic, long-term administration will beindicated in many cases. The dosage may be administered as a single doseor divided into multiple doses. In general, the desired dosage should beadministered at set intervals for a prolonged period, usually at leastover several weeks or months, although longer periods of administrationof several months or years or more may be needed.

The dosage regimen utilizing any agent described herein can be selectedin accordance with a variety of factors including type, species, age,weight, sex and medical condition of the subject; the severity of thecondition to be treated; the route of administration; the renal orhepatic function of the subject; the pharmacogenomic makeup of theindividual; and the specific compound of the invention employed. Anyagent described herein can be administered in a single daily dose, orthe total daily dosage can be administered in divided doses of two,three or four times daily. Furthermore, any agent described herein canbe administered continuously rather than intermittently throughout thedosage regimen.

Methods of Treatment

In various aspects, the present invention provides for a method fortreating or preventing dry AMD and/or RPD. In these aspects, the “agentof the invention” comprise compounds useful for both monotherapy andcombination therapy (e.g. as an additional therapeutic agent). Ingeneral, monotherapy comprises the use of compounds of Formula I,methotrexate, or their pharmaceutically acceptable salts, whilecombination therapy comprises compounds of Formula I, methotrexate, ortheir pharmaceutically acceptable salts in combination with anadditional therapeutic agent, including, one or more of an anti-VEGFagent, an ACE inhibitor, a PPAR-gamma agonist, a renin inhibitor, asteroid, an agent that modulates autophagy PPAR gamma modulator,semapimod, a MIF inhibitor, a CCR2 inhibitor, CKR-2B, a 2-thioimidazole,CAS 445479-97-0, CCX140, clodronate, a clodonate-liposome preparation orgadolinium chloride.

An evaluation of any of the treatments disclosed herein can compriseoptical imaging, including, by way of non-limiting example, cSLO, FAF,OCT (including with cross-sectional, three-dimensional and en faceviewing), SD-OCT (with cross-sectional, three-dimensional and en faceviewing), or other imaging modalities including other wavelengths offluorescence (e.g. wavelengths ranging from blue to infrared, e.g., 390nm to 1 mm, including, for example, blue light, white light, red-free,near infra-red, or infrared).

In some embodiments, the methods described herein comprise reducing theamount of pseudodrusen in the subject and/or reducing the amount ofpseudodrusen in any one of the foveal area, perifoveal area, juxtafovealarea, and extrafoveal area of the subject's eye. In other embodiments,the methods described herein comprise reducing the rates of progressionto late disease, wherein the late disease is any one of choroidalneovascularization or geographic atrophy. In some embodiments, themethods described herein comprise reducing the rates of expansion ofgeographic atrophy.

In some embodiments, the methods of treatment described herein comprisetreatment, prevention, or reduction in the rate of pathogenesis of dryAMD and/or RPD.

Compound Evaluation Methods

In some aspects, the invention provides a method for identifying whethera candidate compound is useful for the treatment of a blinding eyedisease, comprising (a) administering an effective amount of a testcompound to an animal whose eye comprises (i) a fluorescent compound inan amount effective to indicate the presence of a blinding eye diseasein the animal and (ii) a toxin in an amount effective to induce atrophyof ocular tissue; (b) exposing the eye to light having a wavelength andintensity effective to cause the fluorescent compound to fluoresce; (c)comparing the eye's fluorescence pattern to a fluorescence pattern of ananimal's eye that comprises the fluorescent compound and the toxin butnot the test compound; and (d) selecting the test compound as acandidate compound if the result of the comparison of step (c) indicatesthat the test compound is useful for the treatment of a blinding eyedisease. In some embodiments, step (b) comprises exposing the eye tolight having a wavelength and intensity effective to cause thefluorescent compound to fluoresce, whether performed coincidentally withadministration of the fluorescent compound, or later administration ofthe fluorescent compound.

In other embodiments, the comparing occurs at least about 24 hours, orat least about 7 days, or at least about 30 days, or at least 60 days,or at least 90 days after administering the test compound. In otherembodiments, the comparing occurs at least about 2 months, or about 3months, or about 4 months, or about 5 months, or at a maximum about 6months. In some embodiments, the comparing comprises observation of theeye of the same animal pre- and post-administering an effective amountof a test compound. In some embodiments, the comparing comprisesobservation of the eye of different animals under different conditions(e.g. with or without administering an effective amount of a testcompound).

In still other embodiments, the methods further comprise the step ofobserving the eye prior to administering the test compound. In someembodiments, this observing establishes one or more pre-administrationcharacteristics of the eye.

In some embodiments, the comparison and/or observation comprisesevaluating optical imaging, including, by way of non-limiting example,cSLO, FAF, OCT (including with cross-sectional, three-dimensional and enface viewing), SD-OCT (with cross-sectional, three-dimensional and enface viewing), or other imaging modalities including other wavelengthsof fluorescence (e.g. wavelengths ranging from blue to infrared, e.g.,390 nm to 1 mm, including, for example, blue light, white light,red-free, near infra-red, or infrared), between two different conditions(e.g. with or without administering an effective amount of a testcompound).

In some embodiments, a compound is useful for the treatment of ablinding eye disease if it provides treatment, prevention, or reductionin the rate of pathogenesis of a blinding eye disease.

In yet another embodiment, the methods described herein compriseadministering the fluorescent compound prior to administering the testcompound. In still another embodiment, the methods described herein donot comprise administering (i) an additional amount of fluorescentcompound to the animal or (ii) a second fluorescent compound to theanimal.

In other embodiments, the methods described herein compriseadministering the toxin prior to administering the test compound and/oradministering the toxin prior to administering the fluorescent compound.

In other embodiments, a plurality of candidate compounds is identified.In some embodiments, the methods described herein further comprisecomparing the usefulness of the plurality of candidate compounds in thetreatment of a blinding eye disease and selecting a lead compound basedon the comparison. In some embodiments, a lead compound is a preferredcompound among a plurality of candidate compounds.

In some embodiments, the comparison comprises evaluating opticalimaging, including, by way of non-limiting example, cSLO, FAF, OCT(including with cross-sectional, three-dimensional and enface viewing),SD-OCT (with cross-sectional, three-dimensional and enface viewing), orother imaging modalities including other wavelengths of fluorescence(e.g. wavelengths ranging from blue to infrared, e.g., 390 nm to 1 mm,including, for example, blue light, white light, red-free, nearinfra-red, or infrared), between two different conditions (e.g. with afirst candidate compound versus with a second candidate compound). Morethan two candidate compounds can be compared.

Diagnostic and Predictive Methods

In some aspects, the invention provides a method for identifying asubject who has a blinding eye disease and is more likely than not torespond to treatment with an agent comprising determining whether thesubject's eye has, or previously had, an increase (including a transientincrease) in permeability across the epithelial barrier between achoroid and a retina of the eye relative to an undiseased state; whereinthe increase in permeability indicates that the subject is more likelythan not to respond to treatment with the agent; and wherein the agentis selected from methotrexate or a pharmaceutically acceptable saltthereof, or and a compound of Formula I (as described herein) or apharmaceutically acceptable salt thereof, wherein each of R₁ and R₂ isindependently H or a C₁-C₆ alkyl and R₃ is H or a C₁-C₆ alkyl.

In another aspect, the present invention provides a method foridentifying a blinding eye disease subject who is more likely than notto respond to treatment with an agent comprising determining whether thesubject's eye has an presence (e.g. an influx) of phagocytic immunecells across a RPE (and/or from the inner retina), relative to anundiseased state, wherein the presence of phagocytic immune cellsindicates that the subject is more likely than not to respond totreatment with the agent; and wherein the agent is selected frommethotrexate or a pharmaceutically acceptable salt thereof, or and acompound of Formula I (as described herein) or a pharmaceuticallyacceptable salt thereof, wherein each of R₁ and R₂ is independently H ora C₁-C₆ alkyl and R₃ is H or a C₁-C₆ alkyl.

In another aspect, the invention provides a method for determiningwhether a blinding eye disease in a subject is responsive to treatmentwith an agent that inhibits the function of a subject's immune cells,comprising detecting a presence, detecting an absence, or measuring anamount of immune cells in the subject's eye, wherein the subject's eyefluoresces in response to light having a wavelength of about 600 nm toabout 900 nm, or about 400 nm to about 900 nm, or about 400 to about1600 nm.

In some embodiments, the methods described herein further compriseadministering to the subject an effective amount of a fluorescentcompound, wherein the detecting or measuring occurs at least one dayafter the administration of the fluorescent compound. In someembodiments, the detecting or measuring occurs at least one day afteradministering to the subject an effective amount of a fluorescentcompound.

In some embodiments, the methods described herein comprise DNIRA fordetermining whether a blinding eye disease in a subject is responsive totreatment with an agent that inhibits the function of a subject's immunecells.

In some embodiments, the methods described herein further comprise thestep of detecting or measuring FAF in the eye of the subject. In someembodiments, the methods described herein further comprise the step ofcorrelating an FAF pattern to the presence, absence, or amount of immunecells in the subject's eye. In some embodiments, the methods describedherein comprise a correlating between FAF and DNIRA data. In someembodiments, the correlating is of the spatial patterns observed in FAFand the subject's eye fluorescence in response to light having awavelength of about 600 nm to about 900 nm, or about 400 nm to about 900nm, or about 400 to about 1600 nm.

In some embodiments, areas of hyperfluorescent FAF or abnormal patternsof FAF may spatially coincide with areas of abnormal DNIRA, which may behypofluorescent or hyperfluorescent. As abnormal FAF or hyperfluroescentFAF coincides with areas of disease activity and can predict areas ofatrophy, without wishing to be bound by theory, in embodiments in whichDNIRA labels the RPE and spatially coincides with hyperfluorescent FAFor abnormal FAF, phagocytic RPE cells that ingest s dye (for example,ICG) have abnormal amounts of lipofuscin or lipofuscin-like material.Further, without wishing to be bound by theory, in embodiments in whichDNIRA labels immune cells (e.g. phagocytic immune cells, or cells of theinnate immune system), and coincides spatially with hyperfluorescent FAFor abnormal FAF, phagocytic immune cells that ingest a dye (for example,ICG) have abnormal amounts of lipofuscin of lipofuscin-like materialand/or coincide with cells that have abnormal amounts of lipofuscin orlipofuscin-like material. In some embodiments, the co-localization ofabnormal FAF with abnormal DNIRA can therefore identify cellular targetsfor therapy. Such co-localization, provides, in some embodiments, bothin animal models of disease, and in patients, the ability to target theimmune system to reduce, slow, prevent disease progression.

In some embodiments, the detecting or measuring occurs at about one day,or about seven days, or at about thirty days after administration of thefluorescent compound. In other embodiments, the comparing occurs atleast about 2 months, or about 3 months, or about 4 months, or about 5months, or at a maximum about 6 months. In some embodiments, thecomparing comprises observation of the eye of the same animal pre- andpost-administering an effective amount of a test compound. In someembodiments, the comparing comprises observation of the eye of differentanimals under different conditions (e.g. with or without administeringan effective amount of a test compound).

In some embodiments, the comparison comprises evaluating opticalimaging, including, by way of non-limiting example, cSLO, FAF, OCT(including with cross-sectional, three-dimensional and enface viewing),SD-OCT (with cross-sectional, three-dimensional and enface viewing), orother imaging modalities including other wavelengths of fluorescence(e.g. wavelengths ranging from blue to infrared, e.g., 390 nm to 1 mm,including, for example, blue light, white light, red-free, nearinfra-red, or infrared), between two different conditions (e.g. with orwithout administering an effective amount of a test compound).

In some embodiments, the methods described herein do not furthercomprise administering (a) an additional amount of the fluorescentcompound or (b) a second fluorescent compound.

Subjects and/or Animals

In some embodiments, the subject and/or animal is a mammal, e.g., ahuman, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep,or non-human primate, such as a monkey, chimpanzee, or baboon. In otherembodiments, the subject and/or animal is a non-mammal, such, forexample, a zebrafish. In some embodiments, the subject and/or animal maycomprise fluorescently-tagged cells (with e.g. GFP). In someembodiments, the subject and/or animal is a transgenic animal comprisinga fluorescent cell, such as, for example, an RPE cell and/or an immunecell. In some embodiments, the subject and/or animal is a human. In someembodiments, the human is a pediatric human. In other embodiments, thehuman is an adult human. In other embodiments, the human is a geriatrichuman. In other embodiments, the human may be referred to as a patient.

In certain embodiments, the human has an age in a range of from about 0months to about 6 months old, from about 6 to about 12 months old, fromabout 6 to about 18 months old, from about 18 to about 36 months old,from about 1 to about 5 years old, from about 5 to about 10 years old,from about 10 to about 15 years old, from about 15 to about 20 yearsold, from about 20 to about 25 years old, from about 25 to about 30years old, from about 30 to about 35 years old, from about 35 to about40 years old, from about 40 to about 45 years old, from about 45 toabout 50 years old, from about 50 to about 55 years old, from about 55to about 60 years old, from about 60 to about 65 years old, from about65 to about 70 years old, from about 70 to about 75 years old, fromabout 75 to about 80 years old, from about 80 to about 85 years old,from about 85 to about 90 years old, from about 90 to about 95 years oldor from about 95 to about 100 years old.

In other embodiments, the subject is a non-human animal, and thereforethe invention pertains to veterinary use. In a specific embodiment, thenon-human animal is a household pet. In another specific embodiment, thenon-human animal is a livestock animal.

In various embodiments, a subject's and/or an animal's eye comprises (i)a fluorescent compound in an amount effective to indicate the presenceof a blinding eye disease in the subject and/or animal and (ii) a toxinin an amount effective to induce atrophy of ocular tissue. In someembodiments, such a subject and/or animal is administered an agent ofthe invention or is not administered an agent of the invention.

In various embodiments, RPE and immune cells are evaluated and/oreffected. In some embodiments, immune cells include cells of a subject'sand/or animal's innate immune system. In some embodiments, such cellsinclude, but are not limited to, macrophage, monocyte, and microglialcells. In various embodiments, the invention provides for detecting apresence, detecting an absence, or measuring an amount of immune cellsin a subject's and/or animal's eye

Kits

The invention provides kits that can simplify the administration of anyagent described herein. An exemplary kit of the invention comprises anyagent described herein in unit dosage form. In one embodiment, the unitdosage form is a container, such as a pre-filled syringe, which can besterile, containing any agent described herein and a pharmaceuticallyacceptable carrier, diluent, excipient, or vehicle. The kit can furthercomprise a label or printed instructions instructing the use of anyagent described herein. The kit may also include a lid speculum, topicalanesthetic, and a cleaning agent for the ocular surface. The kit canalso further comprise one or more additional agent described herein.

In one embodiment, the kit comprises a container containing an effectiveamount of an agent of the invention, including, for example, compound ofFormula I, methotrexate or a pharmaceutically acceptable salt thereofand an effective amount of another therapeutic agent, such thosedescribed herein.

This invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1: Systemic Injection of the RPE Toxin, NaIO₃, InducesComplex Patterns of FAF Similar to Those of AMD and/or RPD

The RPE toxin, sodium iodate (NaIO₃) generated patchy loss of the RPEand hypofluorescent DNIRA, in the rat eye similar to geographic atrophy(GA). This model not only developed RPE atrophy, it faithfullyreproduced complex patterns of FAF associated with aggressive clinicaldisease, most closely resembling advanced dry AMD, RPD, and the diffusetrickling forms of dry AMD.

Materials: ICG (Cardiogreen), sodium iodate, Harris haematoxylin andeosin, were from Sigma-Aldrich (Oakville, ON, Canada). Tropicamide,0.8%, in 5% phenylephrine hydrochloride solution (Diophenyl-T) was fromSandoz Canada Inc (Boucherville, QC, Canada), and GenTeal lubricatingeye drops were from Novartis Pharmaceuticals Canada Inc (Dorval, QC,Canada). Paraformaldehyde, 32% in phosphate buffered saline (PBS), wasfrom Electron Microscopy Sciences (Hatfield, PA). Mouse anti-rat CD68antibody was from AbD Serotec (Oxford, UK), and mouse IgG was from SantaCruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-Iba-1 was fromWako Pure Chemical Industries Ltd (Osaka, Japan). Alexa-labeledfluorescent goat anti-mouse secondary antibody, Isolectin IB₄ Conjugatesand TO-PRO-3 nucleic acid stain were from Invitrogen (Camarillo, CA,USA). Dako fluorescent mounting medium was from Dako North America(Burlington, ON, Canada).

Animal procedures: all procedures were performed in accordance with theCanadian Council on Animal Care and with adherence to the ARVO Statementfor the Use of Animals in Ophthalmic and Vision Research. For allexperiments, Sprague Dawley (SD) rats aged 6-10 weeks were kept at a 12hour dark/light cycle, with food and water ad libitum. For evaluation,rats were anesthetized with ketamine (100 mg/kg) and xylazine (10mg/kg), and pupils dilated with Diophenyl-T. Eyes were conditioned usingGenTeal lubricating eye drops. Animals were humanely sacrificed byintracardiac injection of T61 (Intervet Canada, Whitby, Canada), eitherafter a single imaging session or after serial imaging.

Sodium iodate (45% solution), was prepared fresh for each set ofexperiments in 0.9% sodium chloride and injected to a finalconcentration of 45 mg/kg body weight. A total of 192 eyes wereevaluated.

Fundus imaging was performed using a commercially available confocalscanning laser ophthalmoscope, (cSLO, Spectralis, Heidelberg RetinalAngiography, HRA-II; Heidelberg Engineering GmbH, Germany), and the EyeExplorer Image Capture system, version 1.1.10. Images were captured at awavelength of 488 nm excitation with 500 nm barrier filter forfluorescein angiography without the barrier filter for red free (RF,green dominant) imaging, at 795/810 nm excitation/emission for ICGangiography, and with a 830 nm laser for infrared reflectance imaging.

To evaluate 488 nm FAF, cSLO imaging was performed in the absence offluorescein dye. Fluorescein was not given at baseline or at any timeduring each study. In cases when it was necessary to simultaneouslyimage the vasculature and provide landmarks for FAF image analysis, ICGwas injected via tail vein, at 2 or 5 mg/kg, into a previously inserted23 gauge tail vein catheter. In a small number of clearly indicatedsituations, such as initial characterization of the model, fluoresceindextran (200 kD) was provided intra-venously. Results from these latterstudies using fluorescein angiography were performed in a subset ofanimals and not included in FAF analysis.

Fundus images were routinely obtained at baseline (day 0, i.e. prior tosystemic NaIO₃ injection), and at days 3 or 4 (3/4), days 7 or 8 (7/8)and days 13 or 14 (13/14) after NaIO₃. In addition, for initialcharacterization of the model, images were acquired at 24 hourspost-NaIO₃, at 21 to 28 days and out to 88 days (3 months). Wherepossible, composite images consisting of at least nine images were takenin the following order: optic nerve head, upper right, right, lowerright, inferior, lower left, left, upper left, and the superior fields.In some cases, a second or third complete or partial ring of images wasobtained more peripherally, typically by realigning the animal relativeto the cSLO as needed. By contrast, in other cases, in which the fullnine images could not be captured, and a partial composite image wasgenerated.

High resolution spectral domain OCT images were obtained (Envisu R2200VHR Animal SDOIS System, Bioptogen, USA). A scans were acquired at arate of 36,000 per second, with a lateral resolution of approximately2.8 μm at the level of the retina.

Tissue Analysis: hemotoxylin and eosin (H & E) histology ofparaffin-embedded tissue was performed as known in the art afterfixation with Davidson's fixative. Paraffin-embedded sections wereviewed with a Nikon Upright E800 Microscope. Deparaffinization andrehydration procedure was followed as in H&E tissue processing.

For immunohistochemistry, sections were blocked in 5% BSA in TBS at roomtemperature and incubated overnight with anti-CD68 antibody at 4° C.with gentle shaking. After rinsing, samples incubated with secondaryantibody for 2 hours at room temperature, and washed again. For negativecontrol, same-species IgG was used at equivalent maximum molarconcentrations as the primary antibody. Nuclei were counter-stained withTO-PRO-3.

Confocal and Fluorescent Microscopy: images were acquired using a TCS SLConfocal fluorescent microscope (Leica Microsystems, GmbH, Wetzlar,Germany), and images captured and analyzed using Leica ConfocalSoftware. Upright epifluorescent microscope (Olympus BX50) was also usedto view the autofluorescent signals of the cells under 488 nm wavelength light. 3D reconstructions of confocal serial z-sections wereperformed using Imaris software version 7.4.2 (Bitplane).

FAF and angiographic imaging in vivo of the normal Sprague Dawley rateye produced a faint, homogenous ground glass glow interrupted byradially-arranged retinal blood vessels that block RPE fluorescence andso appear dark, and by a central hypofluorescent circle at the opticnerve head (ONH), where RPE is absent (FIGS. 1A-D). Three days aftersystemic NaIO₃ injection, a single area of iso- or slightlyhyperfluorescent FAF with hyperfluorescent borders appeared, and tookthe shape of a discrete island, sector, or 360° ring (FIGS. 1A-D).Islands or sectors are consistently located inferior to the ONH or inthe inferior hemiretina (FIGS. 1A-D). When a 360° ring is present, ittypically encompasses much of the fundus, with one border adjacent to,surrounding, the ONH and the other in the mid- to far-retinal periphery.Large 360° rings were the most commonly observed shape, occurring in102/110 (93%) of eyes treated with sodium iodate (FIGS. 2A-D). In allcases, the islands, sectors or rings approached the ONH but were notcontiguous with it (FIGS. 1A-D-3A-C). These and the followingobservations were confirmed in over 160 eyes.

Beginning approximately 4-5 days after NaIO₃ injection, a markedreticular or curvilinear pattern of alternating hyper/hypo-fluorescentFAF appeared within the islands, sectors or rings (FIGS. 1A-D). By day7, this pattern was pronounced. In small lesions, it completely filledthe island or sector and emerged rapidly. In large 360° rings, it was atfirst pronounced at the proximal and distal borders and continued toemerge over the next weeks, whereby its curvilinear componentscontributed to partial loops, ovals, circles, rosettes, and scalloped or“paw-print” patterns of FAF (FIGS. 2A-D). In some regions, curvilinear,round or oval shapes coalesce and appeared darker than the surroundingareas, appearing as homogenously dark grey patches of hypofluorescentFAF. With time, the hyperfluorescent reticular pattern matured in situ,becoming more granular and less smoothly curvilinear (FIGS. 2A-D).However, even as late as 20 weeks, the last time-point analyzed, theborders retained their hyperfluorescent reticular pattern and continuedto creep slightly toward the retinal periphery.

Fluorescein angiography performed in a subset of animals confirmed thatthe reticular pattern does not correspond with the retinal or choroidalvasculature (note that fluorescein-dextran, a 488 nm dye, was not givento animals that underwent FAF imaging, unless indicated) (FIGS. 1A-D).However, ICG angiography revealed a permeability change that occurred atthe chorio-retinal interface, across the RPE and BM, approximately twoto three days after NaIO₃ that coincides with, both spatially andtemporally, the emergence of the islands, sectors of rings of FAF (FIGS.1A-D). This increase in permeability was transient and resolved by Day7, the next timepoint routinely evaluated. The retinal vessels did notleak. In the NaIO₃ model, H&E staining confirms that fluid accumulatesin the subretinal space. This was also observed by ultra-high resolutionOCT in vivo.

To determine the pathological correlate of the FAF pattern observed invivo, excised retinal wholemounts were viewed at low magnification underwhite light and by epifluorescent microscopy at 488 nm—the samewavelength used for in vivo imaging, with no fluorescent IHC labeling.Depending on subsequent tissue analysis, two microscopes were used forthis purpose: an epifluorescent device with a narrow excitation/emissionwindow (488/520 nm), and the Inverted Leica DM IRE2 viewing system of aconfocal microscope with a mercury arc lamp and long-pass (rather thanband-pass) filter providing fluorescence from greater than 515 nmfollowing excitation with a 450-490 nm band. Under white light, thenormal rat retina appears fairly homogenous throughout, with someartifactuous deformation (FIG. 3A). By contrast, a subtle off-white,fine reticular pattern becomes apparent in samples obtained 7 days afterNaIO₃ injection. By day 14, this pattern is more apparent and seen tocorrespond with small, outer retinal folds best illustrated at the cutedge of the retina (FIG. 3B). Viewed under 488 nm fluorescent light,this same curvilinear pattern is apparent. Though apparent by day 7after injection, increasing autofluorescence makes this pattern morevisible by Day 14 (FIG. 3C). Using higher magnification epifluorescentmicroscopy, it was demonstrated that this lacy, curvilinear patterncoincides with the spatial distribution of autofluorescent cells thatappear to align with grooves or folds of the outer retina (FIGS. 3A-C).

FIGS. 4A-C shows a comparison of the clinical features of RPD withtissue findings in the NaIO₃ model. In addition to clinical OCT imagingof the patient with RPD (that shows a base-down pyramidal structure inthe subretinal space), color and red-free fundus images were obtained(FIGS. 4A-C). Red free images showed small dark spots that constituteindividual pseudodrusen of RPD (FIG. 4A, top image). These areidentified using dark grey overlay. We further highlight, circumscribedby circles, the presence of so-called “target lesions” that characterizeRPD. These appear as dark spots with lighter centers. In parallel, aschematized illustration of folded outer rat retina, both en face and incross-section that, when falsely colored and presented as black andwhite images—both “positive” and “negative”—appears to correlatedirectly with target-like lesions, and halos of RPD. Taken together,these data suggest that the 2D patterns of dark pseudodrusen within alacy or reticular pattern of circles, halos and target lesion with darkcenters viewed en face, in 2D, correspond with elevated rings andcraters, considered volumetrically, in 3D. The inter-pseudodrusen matrixconsists of ribbons of persistent subretinal inflammation that whencross-sectioned appear as pyramids or spikes by OCT. The lost outerretina between these structures appears dark. This places theOCT-defined pseudodrusen adjacent to the dark patches of RPD. Withoutwishing to be bound by theory, this correlation with structuraldeformity of the retina further explains why RPD can be visualized inseveral imaging modalities and are not limited to a particularwavelength, such as 488 nm, alone as would occur if the signal werefluorophore dependent.

Example 2: DNIRA of a Rat Eye after Systemic ICG AdministrationIdentifies the Retinal Pigment Epithelial (RPE) Layer In Vivo

It is demonstrated that a near infra-red dye, ICG, can label theRPE/outer retina following systemic injection and so enhance thedetection of RPE and RPE atrophy. The observed change in DNIRA using theICG excitation/emission filters is useful in lieu of FAF in models ofdisease to identify areas of RPE/outer retinal loss.

ICG dye, or PBS, was injected systemically in 46 Sprague Dawley rats atdoses of 0.35 and 5.0 mg/kg, and the fundus was evaluated in vivo usingconfocal scanning laser ophthalmoscopy (cSLO), with 795/810 nmexcitation/emission filters in place, prior to injection, and at days 2,7, and 21 thereafter. Electroretinography (ERG) was performed toevaluate potential toxicity. In a subset of animals, an RPE toxin wasinjected to induce RPE damage or loss.

Specifically, for animal studies, animals were handled in accordancewith the Association for Research in Vision & Ophthalmology (ARVO)guidelines for the humane use of animals in ophthalmic research, andaccording to the St. Michael's Hospital Animal Care Committeeguidelines. 46 Sprague Dawley (SD) rats aged 6-10 weeks, weighing200-300 g were kept on a 12 hour dark/light cycle, with food and waterad libitum. Animals were anesthetized with a combination of ketamine(100 mg/kg) and xylazine (10 mg/kg), and pupils dilated with a singledrop of 0.8% tropicamide in 5% phenylephrine hydrochloride solution(Diophenyl-T, Sandoz Canada Inc). GenTeal lubricating eye drops(Novartis, Canada), were repeatedly applied to the corneal surfaceduring all procedures.

CSLO: in vivo images were acquired using a commercially available cSLO(Heidelberg Retinal Angiography, HRA-2, Heidelberg Engineering,Germany). Images were obtained in the red-free, FAF (488/500 nmexcitation/emission), NIR channel (830 nm) and ICG channel (795/810 nmexcitation/emission).

ICG and Fluorescein Angiography: ICG dye (Cardiogreen, Sigma, Cat#12633) was freshly prepared prior to experimentation to a final stockconcentration of 5.0 mg/ml. A 23-gauge catheter was inserted into thetail vein, and ICG dye infused at doses of 0.35, or 5.0 mg/kg. Imageswere taken prior to injection (baseline), during dye circulation, and atvarious intervals thereafter out to 20 minutes. In a subset of animals,fluorescein-dextran (200 kD) (Sigma, Cat #FD2000S) solution, at 5.0mg/ml, was injected IV via tail vein catheter to yield a final dose of5.0 mg/kg. ICG and fluorescein angiography was performed simultaneouslyin a subset of animals only, otherwise fluorescein was not injected.Angiographic images were obtained in the fluorescein and ICG channelswith excitation and emission filters of 488/500 nm and 795/810 nmrespectively. Control animals received PBS.

DNIRA: DNIRA images were obtained in the days and weeks after ICGinjection using the ICG angiography settings, i.e. withexcitation/emission filters, but without re-injection of dye at 24hours, 48 hours, 3 days, 7 days, 21 days and 28 days after angiography.Angiography was not performed again during the time-course of the study.

Toxin administration: in a subset of animals (n=7), the RPE toxin NaIO₃(Sigma, cat #424064) was injected systemically via a 23 G catheterinserted into the tail vein at a dosage of 45 mg/kg body weight. Inthese animals, ICG angiography at 0.35 mg/kg was performed immediatelyprior to NaIO₃ injection. DNIRA was performed 7 days after ICG and NaIO₃injection

Electroretinography (ERG):animals receiving ICG or PBS were evaluatedusing the Espion (DiagnosysLLC, USA) mini-Ganzfeld ERG system. Followinganesthesia, animals were placed on an electrically silent heating padand gold coil electrodes placed on the corneal surface after applicationof GenTeal lubricating drops. Following a short train of dim flashes(0.01 candela s/m², 1.0 Hz), a scotopic b-wave amplitude was evaluatedusing a single bright flash (3 candela s/m²) that we previouslydetermined consistently produces a response close to the maximum b-waveamplitude. Student t-test was used to compare post-injection amplitudesagainst baseline (prior to injection).

Statistical Analysis Used the ANCOVA Analysis of Regression.

Representative baseline (pre-ICG) cSLO findings in the normal SpragueDawley rat eye are shown (FIGS. 5A-H), and served along with PBS-treatedanimals (FIG. 6A), as normal controls for all experimentation. Red-free(i.e., green dominant) imaging identified the optic nerve head (ONH) andradial blood vessel of the retina (FIG. 5A). Low levels of endogenoussignal were also seen at longer wavelengths, in the NIR channel (830nm), and provided a similar image of the ONH and vasculature with somepotential imaging of the deeper choroidal vessels that arises due todeeper light penetration (FIG. 5B). Consistent with human studies, FAFappeared as a faint ground-glass glow that is lacking in detail, and isobscured by the radial retinal blood vessels and is absent at the ONH(FIG. 5C). This diffuse glow however is very faint.

By contrast, with the NIR excitation/emission filters in place as forICG angiography, no or negligible signal was observed in the eye priorto ICG injection (FIG. 5D). Scan lines were evident and the vasculaturewas barely detectable or not detectable. This was observed in over 60eyes. Immediately following ICG injection, both the retinal andchoroidal vessels (FIG. 5F, and FIG. 5G second panel) were identifiedduring the transit phase and out to the experimental endpoint, 20minutes later. Fluorescein angiography also confirmed the normal retinalvasculature (FIG. 5E). In wild-type SD animals, no leakage was seen fromeither vascular bed using either fluorophore.

Though baseline images prior to ICG angiography exhibited no signalunder NIR stimulation in the presence of the ICG excitation/emissionfilters, images obtained at 3, 8, 21 and 28 days after a singleinjection of ICG at day 0 (t=0), demonstrated a delayed and persistentfluorescence (FIG. 5G). This was not observed in similar experimentsperformed following angiography with fluorescein-dextran dye andanalyzed with the fluorescein angiography filters in place (FIG. 5H),where the same level of low pre-injection fluorescence is visiblethroughout the time course of investigation. It also did not occur inthe absence of previous ICG injection (FIG. 6A). Qualitative analysis ofthis delayed NIR fluorescence after ICG showed a “mosaic” pattern ofsmall speckles in the posterior pole that, viewed enface, are deep(external) to the retinal vessels and obscure the view to the choroidalvasculature, suggesting its location between the retina and the choroid.The ONH and circumpapillary area did not fluoresce. These features aresimilar to those of FAF performed in the blue spectrum in patients. Itwas determined that the same plane of focus is optimal for bothfluorescent techniques.

The observations made using DNIRA after ICG injection weredose-dependent (FIGS. 6A-D). The higher the initial ICG dosage used forangiography, the brighter the NIR signal. The high dose, 5 mg/kg,required that the gain (sensitivity) of the cSLO be reduced to obtainsuitable quality images, while the low dose (0.35 mg/kg) was lessbright, and required that the gain be increased to obtain suitablequality images. Comparative images (FIG. 6A, FIG. 6B and FIG. 6C), takenwith the gain set at the same level, demonstrated a dose-responsiveincrease in brightness. In the same experiment, it was also determinedthat by day 28 the speckled pattern faded in animals receiving low-doseICG (FIG. 6B). By contrast, in animals receiving high dose ICG, therewas little appreciable difference between days 3 and 28 post-injection(FIG. 6C).

NIR reflectance imaging at 830 nm, without 795/810 nmexcitation/emission filters necessary for ICG activation, did not yieldthe speckled, dose-dependent and time-dependent fluorescence findingsobserved with DNIRA.

Having demonstrated that DNIRA identifies the RPE and/or outer retinalcomplex, it was determined that DNIRA could be used to identifystructural abnormalities of the RPE. DNIRA was used in combination withsystemic injection of the RPE toxin, NaIO₃. NaIO₃ is directly toxic toRPE cells and leads to their loss; apoptosis of the overlyingphotoreceptors occurs thereafter. DNIRA in the days and weeks afterNaIO₃ injection identified geographic (spatial) patches of profoundhypofluorescence, evident as large black patches within an otherwisecontinuous background of speckled fluorescence. These hypofluorescentpatches were bounded by a defined border. Further, with the gain of thecSLO increased, brighter viewing through these patches permitted clearvisualization of the choroidal detail (FIGS. 7A-B). Choroidal vesselswere identified by their complexity, variable size, and non-radialpattern with respect to the ONH. By contrast, the speckled layerobscured this view but permitted ongoing visualization of the overlyingretinal vessels. These data were consistent with the notion that DNIRAdetects ICG-labeled RPE/outer retinal layer.

Further, the suitability of DNIRA for pre-clinical experimentation wasdemonstrated with toxicity studies. Electroretinography, comparing highand low dose systemic ICG concentrations against PBS, was undertaken.These data showed no statistically significant change in the b-waveamplitude in the three weeks following injection compared againstchanges noted in control animals, and demonstrate that DNIRA, over thedose range used, is safe in pre-clinical studies of chorioretinaldisease. While the high dose is well above that used clinically, the lowdose is consistent, in grams/body weight, with the dose used forclinical imaging with traditional fundus cameras.

Example 3: Discovery of Compounds for the Treatment of Blinding EyeDiseases

Having established an animal that models a blinding eye disease, a testcompound is administered to such an animal.

The presentation of the blinding eye disease is determined using in vivoimaging including fluorescence detection. A candidate compound isidentified by observing fluorescence in the eye at or after a timesufficient to present characteristics of a blinding eye disease. Thecandidate compound is evaluated for its ability to reduce or eliminatethe characteristics of the blinding eye disease as described herein.

Example 4: Assessing the Activity of Compounds for Blinding Eye Diseases

Having developed an animal that models a blinding eye disease, acandidate compound is administered to such an animal.

The presentation of the blinding eye disease is determined using in vivoimaging including fluorescence detection. The activity of a candidatecompound is assessed by observing fluorescence in the eye at or after atime sufficient to present characteristics of a blinding eye disease andthe activity of a candidate compound. The candidate compound may beassessed for its ability to reduce or eliminate the characteristics ofthe blinding eye disease as described herein.

Example 5: Treatment of Dry AMD with Bindarit

Human subjects, 56 to 100 years of age or more, present with dry AMD, asdiagnosed by one or more of the following clinical tests: clinicalexamination, FAF (at any wavelength), near infrared and/or red-freephotography, fluorescein angiography, which allows for theidentification and localization of abnormal vascular processes; OCT,which provides high-resolution, cross-sectional or en face images fromwithin optical scattering media, such as the human retina and choroid;and structured illumination light microscopy, using a specially designedhigh resolution microscope setup to resolve the fluorescent distributionof small autofluorescent structures (lipofuscin granulae) in retinalpigment epithelium tissue sections.

The subjects are administered bindarit in two 300 mg oral doses once aday for 12 weeks. After an initial twelve-week treatment period, thesubjects are evaluated for clinical outcomes. Alternatively, patientsreceive intravitreal injection of a vehicle containing bindarit, with orwithout a drug delivery vehicle.

A first clinical outcome is determined using a standard visual acuitytest, as is well known in the art. The subjects are assessed for theability to clearly see symbols and objects on a Snellen eye chart from adistance.

A second clinical outcome assesses the rate of progression of geographicatrophy. To do so, the subjects' pupils are dilated with 1.0%tropicamide and 2.5% phenylephrine before retinal imaging. Imaging iscarried out with an instrument (e.g., Spectralis HRA+OCT; HeidelbergEngineering, Heidelberg, Germany) that allows for simultaneous recordingof cSLO and spectral-domain optical coherence tomography (SD-OCT) withtwo independent scanning mirrors, as described in Helb, et al. ActaOphthalmol. 2010 December; 88(8):842-9. Five modes of operation areemployed: white light, red-free light, near infrared, FAF and OCT.

cSLO images are obtained according to a standardized operation protocolthat includes the acquisition of near-infrared reflectance (λ=815 nm)and FAF (excitation at λ=488 nm, emission 500-700 nm) images.Simultaneous SD-OCT imaging is carried out with an illuminationwavelength of 870 nm, an acquisition speed of 40,000 A-scans, and a scandepth of 1.8 mm. Two SD-OCT scans, one vertical and one horizontal, pereye are performed through the approximate foveal center, or in the caseof RPD, in proximity to the vascular arcades of the macula. Fluoresceinangiography (λ=488 nm, emission 500-700 nm, 10% fluorescein dye) isperformed as needed. Color fundus photographs are obtained with a funduscamera (e.g. FF 450 Visupac ZK5; Carl Zeiss Meditec AG, Jena, Germany).

Interpretation of clinical outcome data informs a decision for furthertreatment, if any.

Example 6: Treatment of Dry AMD with a Combination Therapy

Human subjects, 56 to 100 years of age or more, present with dry AMD, asdiagnosed by one or more of the following clinical tests: clinicalexamination, white-light fundus imaging, FAF at any wavelength, nearinfrared and/or red-free photography, blue-light illumination, and/orfluorescein or ICG angiography, which allows for the identification andlocalization of abnormal vascular processes; OCT, which provideshigh-resolution, cross-sectional, three-dimensional and en face imagesfrom within optical scattering media, such as the human retina andchoroid; and structured illumination light microscopy, using a speciallydesigned high resolution microscope or ophthalmoscope set up to resolvethe distribution of small autofluorescent structures (lipofuscin,lipofuscin-like, or other granulae) in retinal pigment epithelium orother cells and cell layers.

The subjects are administered bindarit in two 300 mg oral doses once aday for 12 weeks. The subjects are also administered ranibizumabinjection once per month (roughly 28 days) in a dose of 0.5 mg peraffected eye. After an initial twelve-week treatment period, thesubjects are evaluated for clinical outcomes.

A first clinical outcome is determined using a standard visual acuitytest, as is well known in the art. The subjects are assessed for theability to clearly see symbols and objects on a Snellen eye chart from adistance.

A second clinical outcome assesses the rate of progression of geographicatrophy. To do so, the subjects' pupils are dilated with 1.0%tropicamide and 2.5% phenylephrine or a comparable agent before retinalimaging. Imaging is carried out with an instrument (e.g., SpectralisHRA+OCT; Heidelberg Engineering, Heidelberg, Germany) that allows forsimultaneous recording of cSLO and SD-OCT, as described in Helb, et al.Acta Ophthalmol. 2010 December; 88(8):842-9. Multiple modes of operationcan be employed: white light, red-free light, blue light, near infrared,and OCT. Similar analysis can be performed with a modified funduscamera.

cSLO images are obtained according to protocols known in the art thatmay include the acquisition of near-infrared reflectance (λ=800-1000 nm)and FAF (excitation at λ=280-550 nm, emission 350-700 nm) images.Simultaneous SD-OCT imaging is carried out with an, for example,illumination wavelength of 870 nm, an acquisition speed of 40,000A-scans, and a scan depth of 1.8 mm. Multiple SD-OCT scans per eye areperformed through the macula and additionally or in the case of RPD, inproximity to the vascular arcades of the macula. Other OCT imaging, suchas, for example, time domain and swept domain, can also be used.Fluorescein angiography (λ=488 nm, emission 500-700 nm, 10% fluoresceindye) is performed as needed. Color fundus photographs are obtained witha fundus camera (e.g. FF 450 Visupac ZK5; Carl Zeiss Meditec AG, Jena,Germany).

Interpretation of clinical outcome data informs a decision for furthertreatment, if any. Illustrative data analysis includes macular cubeanalysis and 5 line raster.

Example 7: Detection and/or Prediction of a Blinding Eye Disease SubjectResponse to an Agent

Using multi-modal imaging in the rat eye, it was determined thatsystemic injection of the RPE toxin, NaIO₃, induces complex patterns ofFAF similar to those in patients with aggressive forms of dry AMD and/orRPD. Tissue histology and fluorescent microscopy illustrated that the invivo patterns of FAF correspond with the spatial distribution ofautofluorescent cells of the innate immune system recruited to areas ofRPE damage or loss.

Multi-modal and Angiographic Imaging In Vivo: Fundus imaging wasperformed using a commercially available confocal scanning laserophthalmoscope, (cSLO, Spectralis, Heidelberg Retinal Angiography,HRA-II; Heidelberg Engineering GmbH, Germany), and the Eye ExplorerImage Capture system, version 1.1.10. Images were captured at awavelength of 488 nm excitation with 500 nm barrier filter forfluorescein angiography without the barrier filter for red free (RF,green dominant) imaging, at 795/810 nm excitation/emission for ICGangiography, and with a 830 nm laser for infrared reflectance imaging.To evaluate 488 nm FAF, cSLO imaging was performed in the absence offluorescein dye. In cases in which it was necessary to simultaneouslyimage the vasculature and provide landmarks for FAF image analysis, ICGwas injected via tail vein, at 2 or 5 mg/kg, into a previously inserted23 gauge tail vein catheter.

FAF imaging of normal Sprague Dawley rat eye produced a faint,homogenous ground glass glow interrupted by radially-arranged retinalblood vessels that block RPE fluorescence and therefore appear dark.Such imaging was also characterized by a central hypofluorescent circleat the optic nerve head (ONH), where RPE is absent. Systemic injectionsof NaIO₃ (45% solution, prepared fresh for each set of experiments in0.9% sodium chloride and injected to a final concentration of 45 mg/kgbody weight) were given to experimental rats. Three days afterinjection, a single area of iso- or slightly hyperfluorescent FAF withhyperfluorescent borders appeared and took the shape of a discreteisland, sector, or 3600 ring. In all cases, the islands, sectors orrings approached the ONH but were not contiguous with it. These and thefollowing observations were confirmed in over 160 eyes.

Beginning approximately 4 to 5 days after NaIO₃ injection, a markedreticular or curvilinear pattern of alternating hyper/hypo-fluorescentFAF appeared within the islands, sectors, or rings. By day 7, thispattern was pronounced and it continued to emerge over weeks, wherebyits curvilinear components contributed to partial loops, ovals, circles,rosettes, and scalloped or “paw-print” patterns of FAF. Even as late as20 weeks, the last time-point analyzed, the borders retained theirhyperfluorescent reticular pattern and continued to creep slightlytoward the retinal periphery.

Fluorescein angiography performed in a subset of animals confirmed thatthe reticular pattern does not correspond with the retinal or choroidalvasculature while ICG angiography revealed a permeability change thatoccurred at the chorio-retinal interface, across the RPE and BM,approximately two to three days after NaIO₃ that coincides with, bothspatially and temporally, the emergence of the islands, sectors of ringsof FAF. This increase in permeability is transient and resolved by day7, the next time point routinely evaluated. The retinal vessels do notleak. Hemotoxylin and eosin (H & E) histology of paraffin-embeddedtissue and immunohistochemistry (IHC) (known in the art; performed afterfixation with, e.g., Davidson's fixative and paraffin-embedded sections,viewed with a Nikon Upright E800 Microscope) confirmed that fluidaccumulated in the subretinal space. This was also observed byultra-high resolution OCT in vivo (high resolution 160 nm spectraldomain OCT images were obtained using the Envisu R2200 VHR Animal SDOISSystem, Bioptogen, USA; scans were acquired at a rate of 36,000 persecond, with a lateral resolution of approximately 2.8 μm at the levelof the retina).

Autofluorescent and Immunofluorescent Analysis of Excised Retina andRPE: To determine the pathological correlate of the FAF pattern observedin vivo, excised retinal whole mounts were viewed at low magnificationunder white light and by epifluorescent microscopy at 488 nm, the samewavelength used for in vivo imaging, with no fluorescent IHC labeling.Depending on subsequent tissue analysis, two microscopes were used forthis purpose: an epifluorescent device with a narrow excitation/emissionwindow (488/520 nm), and the Inverted Leica DM IRE2 viewing system of aconfocal microscope with a mercury arc lamp and long-pass (rather thanband-pass) filter providing fluorescence from greater than 515 nmfollowing excitation with a 450-490 nm band. Under white light, thenormal rat retina appeared fairly homogenous throughout, with someartifactuous deformation. By contrast, a subtle off-white, finereticular pattern became apparent in samples obtained 7 days after NaIO₃injection. By day 14, this pattern was more apparent and was observed tocorrespond with small, outer retinal folds best illustrated at the cutedge of the retina. Viewed under 488 nm fluorescent light, this samecurvilinear pattern was apparent. Using higher magnificationepifluorescent microscopy, it was demonstrated that this lacy,curvilinear pattern coincides with the spatial distribution ofautofluorescent cells that appear to align with grooves or folds of theouter retina.

To determine the identity of the autofluorescent cells that coincidewith the reticular pattern of FAF, excised retina were stained for withmarkers of the innate immune system, avoiding the 488 nm channel. Iba1is a pan-microglial/macrophage marker and in the rat retina; antibodiesagainst it identify the major phagocytic cell populations. Using thismarker, it was found that Iba1⁺ cells are arranged in a lacy,curvilinear pattern when viewed enface in whole mount retina, i.e. inthe z-plane. Further, when compared against photoreceptor nuclearstaining, this curvilinear pattern is seen to lie interposed betweenfolds of the deformed ONL. This pattern of Iba1⁺ cells corresponds withthe pattern of hyperfluorescence observed in vivo by FAF imaging, andwith the pattern of autofluorescent cells in excised retina.Corresponding patterns of in vivo FAF and Iba1⁺ staining were observedat both 7 days and 12 weeks after NaIO₃ injection. Importantly, at 7days after NaIO₃, all RPE cells are absent from the posterior eye cup,confirming that these cells cannot be responsible for the observed FAF.Also, identification of the autofluorescent cells as phagocytic wasconfirmed by comparison of imaging to preparations in whichmonocyte/macrophage depletion has been undertaken. Such depletion wasachieved by treatment with gadolinium chloride (GAD). Upon treatmentwith GAD, far less well defined reticular patterns of FAF were observed,including a reduction in demarcation of the borders.

Such identification of phagocytic cells is indicative of dry AMD and/orRPD and predictive of a subject's response to an agent. Specifically,the observation of such phagocytic cells makes it more likely than notthat a subject will respond to treatment with an agent.

Identification of a transient increase in permeability across asubject's epithelial barrier between a choroid and a retina relative toan undiseased state is indicative of dry AMD and/or RPD and predictiveof a subject's response to an agent. Specifically, the observation of atransient increase in permeability across a subject's epithelial barrierbetween a choroid and a retina relative to an undiseased state makes itmore likely than not that a subject will respond to treatment with anagent.

Delayed Near InfraRed Analysis (DNIRA) of Excised Retina and RPE: Also,identification of abnormal features in comparison with the normal eye isundertaken with Delayed Near Infra Red Analysis (DNIRA). DNIRA imagesare obtained in the days and weeks after dye injection, such as ICGinjection, using the ICG angiography settings, i.e. withexcitation/emission filters, but without re-injection of dye at 24hours, 48 hours, 3 days, 7 days, 21 days or 28 days after angiography.Angiography is not performed again during the time-course of the study.

For toxin administration, a toxin such as NaIO₃ (Sigma, cat #424064) isinjected systemically via a 23 G catheter at a dosage of 45 mg/kg bodyweight. ICG angiography at 0.35 mg/kg is performed immediately prior toNaIO₃ injection. DNIRA is performed 7 days after ICG and NaIO₃injection.

Example 8: DNIRA of a Rat Eye after Systemic ICG Administration LabelsImmune Cells in Vivo

To determine the pathological correlate of the FAF pattern observed invivo, excised retinal wholemounts were viewed at low magnification underwhite light and by epifluorescent microscopy at 488 nm—the samewavelength used for in vivo imaging, with no fluorescent IHC labeling.Depending on subsequent tissue analysis, two microscopes were used forthis purpose: an epifluorescent device with a narrow excitation/emissionwindow (488/520 nm), and the Inverted Leica DM IRE2 viewing system of aconfocal microscope with a mercury arc lamp and long-pass (rather thanband-pass) filter providing fluorescence from greater than 515 nmfollowing excitation with a 450-490 nm band. Under white light, thenormal rat retina appeared fairly homogenous throughout, with someartifactuous deformation (FIG. 3A). By contrast, a subtle off-white,fine reticular pattern became clear in samples obtained 7 days afterNaIO₃ injection. By day 14, this pattern was more clear and seen tocorrespond with small, outer retinal folds best illustrated at the cutedge of the retina (FIG. 3B). Viewed under 488 nm fluorescent light,this same curvilinear pattern was apparent. Though clear by day 7 afterinjection, increasing autofluorescence made this pattern more visible byDay 14 (FIG. 3C). Using higher magnification epifluorescent microscopy,we demonstrated for the first time that this lacy, curvilinear patterncoincides with the spatial distribution of autofluorescent cells thatappear to align with grooves or folds of the outer retina (FIG. 3D).

Without wishing to be bound by theory, this suggests that the in vivopattern of FAF observed corresponds with distribution of autofluorescentcells in the outer retina. This contrasts with the prevailing theoriesthat ascribe clinically-relevant patterns of FAF to abnormal RPE, notingthat RPE are absent in the isolated retinal samples we used. Withoutwishing to be bound by theory, this also suggests that the 2-dimensionalpattern of FAF observed in this model corresponds with distribution ofautofluorescent cells confined within 3-dimensional curvilinear folds ofthe outer retina.

Accordingly, the spatial and temporal changes of the outer retinalstructure and the identity of the autofluorescent cell types wasinvestigated.

Owing to the laminar nature of the neuroretina, we reasoned that outerretinal deformation could be readily evaluated by observingconformational changes of the normally flat outer nuclear layer (ONL), alayer of densely arrayed photoreceptor nuclei. Therefore, immediatelyafter autofluorescent analysis of freshly excised wholemount retina,nuclear staining was performed and the tissue evaluated byepifluorescent or confocal microscopy in a non-488 channel.Cross-sections were also evaluated by H&E of whole eyes.

At baseline prior to NaIO₃ administration, confocal microscopy using thenuclear stain Topro3 confirms that the ONL was flat and devoid ofvascular staining (FIG. 4C). Three days after NaIO₃ administration,slight curvilinear grooves of the ONL were noted, accompanied byoccasional shallow, linear creases. By Day 7, ONL deformation was morepronounced, producing a distinctly interconnected series of grooves. ByDay 14 these changes were more complex and of higher amplitude.

Cross-sectional H & E histology of retina at 900 to images obtainedusing confocal microscopy, showed that NaIO₃-induced RPE toxicity firstleads to small undulations of the ONL that accompany increasingsubretinal fluid, with little photoreceptor loss (FIG. 4B). Later, frankphotoreceptor loss created higher amplitude folds in which the troughsof the ONL are juxtaposed against Bruch's membrane (BM), the basementmembrane of the RPE. Eventually, lateral expansion of these troughs ledto large contiguous regions of outer retinal degeneration such that muchof the inner retina lies juxtaposed against BM.

To determine the identity of the autofluorescent cells that coincidewith the reticular pattern of FAF, the excised retina with markers ofthe innate immune system was statined, avoiding the 488 nm channel. Iba1is a pan-microglial/macrophage marker and in the rat retina; antibodiesagainst it identify the major phagocytic cell populations. Using thismarker, it was shown that Iba1⁺ cells are arranged in a lacy,curvilinear pattern when viewed en face in wholemount retina, namely, inthe z-plane (FIGS. 5A-H). Further, when compared against photoreceptornuclear staining, this curvilinear pattern was seen to lie interposedbetween folds of the deformed ONL. As shown in FIGS. 5A-H, this patternof Iba1⁺ cells corresponded with the pattern of hyperfluorescenceobserved in vivo by FAF imaging (FIGS. 1A-D and 2A-D), and with thepattern of autofluorescent cells in excised retina (FIGS. 3A-C).Corresponding patterns of in vivo FAF and Iba1⁺ staining are shown atboth 7 days and 12 weeks after NaIO₃ injection (FIGS. 5A and 5B). By 7days after NaIO₃ administration, all RPE cells were absent from theposterior eye cup, confirming that these cells were not responsible forthe observed FAF (FIGS. 5C and 5D).

The 3-dimensional (3D) relationship between the Iba1⁺ cells and theouter retina was next evaluated by en face serial confocal microscopy ofwholemount retina (i.e., in the z-axis) moving step-wise from the innerplexiform layer (INL) through the outer plexiform layer (OPL), to theinner-, mid- and outer-ONL. Short stacks of confocal images (segments)were projected (flattened) at these three steps (FIGS. 6A-D). Theco-distribution of activated CD68⁺ macrophages of the monocyte lineagewas also evaluated.

At baseline prior to NaIO₃ administration, Iba1⁺ cells were seen in theIPL only. No CD68⁺ cells were identified (FIG. 6A). Three to four daysafter NaIO₃ administration, Iba1⁺ cells increased in number and wereincreased in the IPL and also throughout the outer retina, including theouter-ONL (FIG. 6B). Two weeks after NaIO₃ administration, a complexdistribution of Iba1⁺ and CD68⁺ cells was seen interlaced betweencurvilinear folds of the ONL. Further, well-defined optically-sectionedectopic circles or ovals of photoreceptor nuclei were observed as farinternally as the OPL, the mid-retinal layer of blood vessels, therebybringing the deformed photoreceptor nuclear layer and vascular layer,two normally separate tissue compartments, in contact. In the mid-ONL,ovoid cross-sections of the deformed nuclear layer appeared morecurvilinear or pisciform, and large Iba1⁺ and CD68⁺ cells were foundwithin their central void. In the outer-most third of the ONL, opticalcross-sections remain curvilinear and were largely contiguous with manybridging segments. The subretinal space, normally a potential spaceonly, was expanded by outer retinal deformation and shows Iba1⁺ and/orCD68⁺ inflammatory cells and photoreceptor nuclei together immediatelyinternal to Bruch's membrane (in the space where the lost photoreceptorouter segments would normally reside).

Confocal scanning microscopy and three-dimensional reconstruction of theexcised wholemount retina was undertaken (FIG. 7A). These data showedthat Iba1⁺ cells formed a reticular or lacy pattern that is interposedbetween photoreceptor nuclei. Further, projection of the z-stack,divided into the same three planes (FIG. 7B right) showed that theinner-ONL is characterized by circular or oval cross-sections of theONL. By contrast, the mid-ONL is characterized by looping curvedcross-sections of the nuclear layer, and the outer ONL is formed oflarger curvilinear segments with multiple bridging elements. Obliquevolumetric projections of these layers readily illustrate the resultingpseudo-egg-crate configuration of the ONL, with several conical peaksextending from single contiguous folds at its base (FIG. 7B middle andright). Without wishing to be bound by theory, these data demonstratethat the majority of inflammatory cells are not located within orbetween ONL troughs, but rather are found under (i.e. external to) theONL peaks in the expanded subretinal space.

Without wishing to be bound by theory, these observations show that the3-dimensional distribution of autofluorescent inflammatory cells underthe deformed outer retina accounts for the 2D pattern of in vivo FAFimaging in these animals.

To correlate these observations made in excised tissue to in vivo changeand FAF findings, 3D volumetric analysis of the living retina usinghigh-resolution OCT in vivo was performed. HR-OCT provides non-invasive,in vivo cross-sections of the retina previously observed only inpost-enucleation histological specimens. OCT imaging is dependent on thesignal generated at the interface between layers of differing opticaldensity, and in the normal rat retina confirms its multi-laminarstructure. The three major outermost OCT boundaries of the retina are,from internal to external, the external limiting membrane (ELM), thejunction of photoreceptor inner and outer segments (IS/OS), and theRPE/BM (FIG. 8A, left). Standard techniques both in the clinic and inthese investigations arbitrarily set the nuclear layers, including theONL, as dark (i.e. a signal void).

Three days after NaIO₃ administration, a reconstructed en face image ofthe living retina captured using OCT demonstrated a subtle change thatis limited to the region inferior to the optic nerve (FIG. 8A, middle).Compared against baseline and the superior retina (which appears grosslynormal three days after NaIO₃ administration) (FIG. 8A, middle), opticalcross sections in the inferior retina showed slight deformation of thephotoreceptor IS/OS junction and ELM such that bright (hyper-echoic)shallow mounds become visible (FIG. 8A, right). This signal was foundexternal to the ONL and internal to BM. The fine dark line representingthat the RPE was no longer present. By day 14, these mounds matured intopronounced peaks, a portion of which form bright, distinct triangles orpyramids whose bases appear to sit on BM (FIGS. 8B and 8C). Other moundsformed discrete narrow spikes that extended from the ELM, through thephotoreceptor layer to reach the mid-retina. A clinical OCT image from ahuman patient with RPD shows a pyramidal subretinal deposit that ispathognomic of this disease (FIG. 8D).

To test if the bright mounds, triangles and spikes that characterize RPDand that seen in the present system, correspond with the distribution ofautofluorescent phagocytic cells observed in the sub-retinal spacevolume-intensity projections (VIPs) were used. These were composed ofOCT-generated optical sections to provide a means of evaluating3-dimensional blocks of living tissue in vivo that can be reconstructedin the z- and y-axis.

Reconstruction of adjacent OCT images in the y-axis showed that themounds or triangles, arrayed side-by-side, contribute to complexstructures such as circles, ovals, halos and target-like lesions. In theexample shown in FIG. 9A, two subretinal mounds form the “sides” of acircle and lie close together at its superior and inferior aspect andmaximally apart in the middle. Single mounds, pyramids and spikescontribute to simpler curvilinear structures.

To further support this finding, VIPs in the z-axis, (that is, instep-wise short segments from the inner-ONL to outer-ONL), were thenanalyzed (FIG. 9B). VIPs through the inner-ONL showed a series ofpunctate spots, regularly spaced throughout the retina. VIPs from themid-ONL showed less regularly spaced pisciform or curved short lines,some of which are bridging. VIPs from the outer-ONL showed contiguouscurvilinear segments with many bridging elements. These findingsdirectly correspond with the 3D data generated from excised wholemountretina (FIGS. 6A-D).

Further, to confirm that hyperfluorescent FAF or abnormal patterns ofFAF and hyperfluorescent DNIRA correlated or co-localized with thedistribution of phagocytic cells, the circulating macrophage/monocytepopulation was depleted using gadolinium chloride (GAD or GdCl₃), a rareearth metal salt which depresses macrophage activity. GAD depletion ofimmune cells led to profound alteration of the NaIO₃-induced pattern ofFAF and DNIRA. GAD depletion of the monocyte/macrophage populations ledto profound alteration of the NaIO₃-induced pattern of FAF. This wasevident qualitatively both within the geographic areas of damage, and atits border (FIGS. 8A-C).

Example 9: Clinical In Vivo Imaging of RPD and Diffuse Trickling AMD

In FIGS. 10A-C, images and interpretive diagrams that directly comparethe clinical features of RPD against tissue findings in the NaIO₃ modelare provided. In addition to clinical OCT imaging of a human patientwith RPD (that shows a base-down pyramidal structure in the subretinalspace, FIG. 8D), color and red-free fundus images were also obtained(FIGS. 10A-C). Red free images showed small dark spots that constituteindividual pseudodrusen of RPD (FIG. 10A, top image). These wereidentified using dark grey overlay. This further highlights,circumscribed by circles, the presence of so-called “target lesions”that characterize RPD. These appear as dark spots with lighter centers.In parallel, a schematized illustration of folded outer rat retina ispresented, both enface and in cross-section that, when falsely coloredand presented as black and white images-both “positive” and“negative”-correlates directly with targets and halos of RPD.

These data indicate that the 2D patterns of dark pseudodrusen within alacy or reticular pattern of circles, halos and target lesion with darkcenters viewed en face, in 2D, correspond with elevated rings andcraters, considered volumetrically, in 3D.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporatedby reference in their entireties.

1.-86. (canceled)
 87. A method for treating dry age related maculardegeneration (AMD) or reticular pseudodrusen (RPD) disease, comprisingadministering to a subject in need thereof an effective amount of apharmaceutical composition comprising a compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein: each of R₁ andR₂ is independently H or a C1-C6 alkyl and R₃ is H or a C1-C6 alkyl. 88.The method of claim 87, wherein the compound of Formula I is bindarit.89. The method of claim 87, wherein the dry AMD is early stage AMD oratrophic AMD.
 90. The method of claim 87, wherein the method reduces therate of progression to late disease.
 91. The method of claim 87, whereinthe method reduces the rate of expansion of geographic atrophy.
 92. Themethod of claim 87, wherein the method reduces the onset of geographicatrophy.
 93. The method of claim 92, wherein the onset of geographicatrophy in the subject is assessed using optical coherence tomography(OCT).
 94. The method of claim 87, wherein the method comprises reducingthe amount of pseudodrusen in the subject.
 95. The method of claim 94,wherein the method comprises reducing the amount of pseudodrusen in anyone of the foveal area, perifoveal area, and juxtafoveal area of thesubject's eye.
 96. The method of claim 94, wherein the reduction in theamount of pseudodrusen in the subject is assessed using opticalcoherence tomography (OCT).
 97. The method of claim 87, wherein themethod comprises reducing expansion of pseudodrusen in the subject. 98.The method of claim 97, wherein the method comprises reducing expansionof pseudodrusen in any one of the foveal area, perifoveal area, andjuxtafoveal area of the subject's eye.
 99. The method of claim 87,wherein the method further comprises administering an additionaltherapeutic agent.
 100. The method of claim 87, wherein thepharmaceutical composition is formulated for sustained release.
 101. Amethod for reducing the rate of expansion of geographic atrophy,progression to late AMD, or onset of atrophy in an eye of a subjectafflicted with dry age related macular degeneration (AMD), comprisingadministering to a subject in need thereof an effective amount of apharmaceutical composition comprising a compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein: each of R₁ andR₂ is independently H or a C1-C6 alkyl and R₃ is H or a C1-C6 alkyl.102. The method of claim 101, wherein the compound of Formula I isbindarit.
 103. The method of claim 102, wherein the pharmaceuticalcomposition is formulated for sustained release.